風疹病毒包膜糖蛋白細胞融合活性位點的定位及衣殼蛋白對融合活性的影響
發(fā)布時間：2018-08-06 09:29
【摘要】： 風疹病毒(rubella virus,RV)是披膜病毒科風疹病毒屬的唯一成員,人類是RV的唯一自然宿主。RV自然感染僅引起輕微的臨床癥狀,許多感染是無癥狀的亞臨床感染。一般于感染后16～20天內出現(xiàn)皮疹,首先于面部出現(xiàn),然后擴散到軀干及四肢,其余癥狀還包括低熱、淋巴結腫大和咽痛。RV感染的并發(fā)癥以關節(jié)炎和關節(jié)痛最為常見,而且多發(fā)生于婦女。 RV引起的主要問題是它的致畸性,即母親孕期感染RV會導致胎兒發(fā)生先天性風疹綜合征(congenital rubella syndrome,CRS)。CRS的臨床表現(xiàn)多種多樣,其中耳聾最為常見,還包括心臟疾病、精神發(fā)育遲滯和眼部疾患如白內障和青光眼。妊娠期間母親感染RV越早,胎兒損害越嚴重。 成熟的RV病毒顆粒是直徑為60nm的球形,核心是衣殼蛋白和單股正鏈基因組40S RNA組成的核衣殼,其外包繞脂質雙層膜,膜上是長度為5～6nm的刺突——由糖蛋白E2和E1組成。衣殼蛋白(capsid protein,CP)是非糖基化的磷酸蛋白,靠二硫鍵形成同源二聚體。CP富含脯氨酸和精氨酸,與RV基因組RNA的結合有關。 包膜糖蛋白E1和E2都是Ⅰ型膜糖蛋白,在病毒表面以異二聚體的形式存在。E1和E2都含有一段跨膜區(qū)(TM),長度分別為22和39個氨基酸。在E2中,TM之后是一段帶正電荷的七氨基酸序列和E1的20個氨基酸信號肽。E1和E2都富含半胱氨酸殘基,E1胞外功能區(qū)的20個半胱氨酸都形成二硫鍵,E2總共含有14個半胱氨酸殘基。E1有3個N-聯(lián)糖基化位點,E2的N-聯(lián)糖基化位點數(shù)目在不同的毒株間有差別,除N-聯(lián)糖基化位點外,E2還含有O-聯(lián)糖基化位點。 E1和E2的功能研究較為廣泛,單克隆抗體研究發(fā)現(xiàn)E1至少含有6個不重復的抗原位點,與血凝和中和活性有關。E1還與病毒和細胞的吸附有關,是主要的表面蛋白。E2的功能研究顯得較為困難,因為它與E1結合后,幾乎不暴露在細胞表面,也就無法用單克隆抗體識別其抗原位點。但是,E2也含有部分血凝表位和中和表位,還有株特異性表位。 細胞融合是包括RV在內的許多有膜病毒侵入細胞、復制、釋放、傳播、致病的重要生物過程,也是細胞間信息傳遞的重要步驟。RV入侵細胞的途徑還沒有弄清楚,但是有證據表明是通過內吞途徑進入細胞。Katow和Sugiura發(fā)現(xiàn)pH在6.0以下時,E1和E2會發(fā)生構象改變,這種改變有利于病毒包膜與內含體膜的融合。 若能弄清RV引起細胞融合的機制,從而改變RV的基因組,改變它的表達產物和生物學活性,就可以消除或降低RV的致畸性;也可以通過改變其侵入細胞或復制的特性來消除或減少胎兒感染的危險性;還可以為研制更安全有效的RV新型基因工程疫苗(基因缺失活疫苗、蛋白工程疫苗等)和特異性抗病毒的多肽類藥物奠定基礎。 細胞融合由位于細胞表面的蛋白引起,因此細胞融合功能的檢測需要所研究的蛋白能夠在細胞表面表達。RV E2的信號肽在病毒結構蛋白的加工及轉運過程中發(fā)揮重要作用,為了使所表達蛋白能夠在細胞內正確加工及順利轉運至細胞表面,本研究構建了重組質粒pBSK-SPE2E1,即將E2的信號肽序列、E2和E1的全基因序列克隆到載體pBluescriptⅡSK~+的EcoRⅠ和XbaⅠ酶切位點之間。然后利用定點突變和同源重組的方法,構建一系列突變體,Giemsa染色和指示基因法檢測它們的細胞融合活性變化,流式細胞術(FACS)檢測蛋白在細胞表面表達效率,Western blot檢測總表達量的改變,血吸附實驗檢測受體識別活性,分析突變位點對RV包膜糖蛋白細胞融合活性的影響,以確定具有細胞融合活性的位點。本研究還構建了RV CP的重組載體pBSK-C,并檢測CP對包膜糖蛋白細胞融合活性的影響。 一、E1胞外功能區(qū)二硫鍵對RV細胞融合活性的影響 RV E1包膜糖蛋白胞外功能區(qū)含有20個半胱氨酸殘基,而且都形成分子內二硫鍵。本研究利用定點突變和同源重組相結合的方法,將RV JR23株E1蛋白胞外功能區(qū)的20個半胱氨酸中的11個突變?yōu)槠渌被?構建了11個突變體Cys2、Cys3、Cys4、Cys5、Cys6、Cys8、Cys9、Cys12、Cys13、Cys17和Cys20,每個突變體去除E1的1個二硫鍵,檢測單個二硫鍵的消失對E1細胞融合活性的影響。 Western blot結果顯示所有突變體蛋白在細胞內的總表達量都沒有降低,但是Cys5和Cys8在細胞表面表達量劇烈下降,所以二硫鍵C(5)-C(8)可以影響E1和E2的相互作用,使它們不能正確地形成異二聚體,從而不能順利轉運到細胞表面。突變體Cys2、Cys6、Cys9、Cys12、Cys17和Cys20的E1、E2蛋白細胞表面表達效率與野毒株相比有所下降,所以這些半胱氨酸形成的二硫鍵可能影響E1、E2的相互作用,也可能影響它們在細胞內的轉運。突變體Cys3、Cys4和Cys13中,E1和E2蛋白的表達效率分別為野毒株的121%、107%和114%,因此半胱氨酸C(3)、C(4)和C(13)形成的二硫鍵對包膜糖蛋白的相互作用及在細胞內的轉運沒有影響。 盡管11個突變體中大部分都有一定數(shù)量的突變蛋白表達在細胞表面,但是所有突變體轉染的細胞中都沒有檢測到多核巨細胞,所以E1胞外功能區(qū)的10個二硫鍵都對RV的細胞融合活性有重要作用。 二、E2中半胱氨酸對RV細胞融合活性的影響 RV包膜糖蛋白E2中含有14個半胱氨酸,其中12個位于胞外功能區(qū),1個位于跨膜區(qū),1個位于胞質區(qū)。本研究利用定點突變和體內同源重組的方法,用突變的寡核苷酸為引物,構建了14個E2的半胱氨酸突變體,每個突變體去除一個半胱氨酸,這些突變體分別為C69T、C82S、C91S、C124G、C132A、C139P、C152G、C157R、C172A、C196G、C207G、C219T、C255W和C259G。 Western blot表明,E2的14個半胱氨酸殘基中,第132位和219位半胱氨酸的改變會導致E1蛋白總表達量降低,其細胞融合活性的降低可能與此有關。其余12位半胱氨酸的改變不影響E1蛋白總表達量,其中C69T、C82S、C124G、C132A、C139P、C152G、C157R、C172A、C196G、C207G、C219T和C255W 10個突變體幾乎喪失細胞融合活性,說明在這些突變體中,E1不能有效運輸?shù)郊毎砻?或者雖然能夠到達細胞表面,但是構象改變,從而不能有效暴露融合活性位點。 突變體C91S和C259G的細胞融合活性與野毒株相近,說明E2第91和259位半胱氨酸的改變不影響E1、E2的相互作用,從而使E1能有效轉運到細胞表面發(fā)揮其細胞融合作用。 實驗中所用的多克隆抗體檢測不到突變的E2蛋白,可見E2的半胱氨酸殘基對維持蛋白的構象有重要作用,半胱氨酸的去除直接導致抗原性的消失,所以推測E2的半胱氨酸可能大部分形成二硫鍵,從而間接影響RV的細胞融合活性。 三、E1關鍵氨基酸突變體對細胞融合活性的影響 E1胞外功能區(qū)的半胱氨酸突變分析顯示,第3、4和13位半胱氨酸突變之后,在細胞表面表達量與野毒株相比沒有降低,但是卻檢測不到融合活性,這3個半胱氨酸所形成的二硫鍵集中在E1的213～285位氨基酸之間,而這一段區(qū)域富含RV中和表位和血凝抑制表位,具有較重要的生物學活性,我們在此區(qū)域選擇了一些保守的或結構上比較特殊的氨基酸,構建了12個突變體H226Q、H238Q、R252S、P253T、R254Q、R256T、L257T、D259G、D261G、P263A、R266Q和P269S。 將各突變體質粒轉染BHK21細胞,24h后Giemsa染色定性檢測各突變體蛋白引起的細胞融合情況,發(fā)現(xiàn)突變體H226Q、R252S和R254Q引起的細胞融合程度與野毒株相近,而突變體R256T和P263A也能引起細胞融合,但是細胞融合強度與野毒株相比有所降低,突變體H238Q、P253T、L257T、D259G、D261G、R266Q和P269S只引起很輕微的細胞融合甚至不能引起融合。 四、衣殼蛋白CP對RV包膜糖蛋白細胞融合活性的影響 RV衣殼蛋白CP在病毒復制、組裝及感染過程中都有作用,本研究檢測其對包膜糖蛋白的細胞融合活性的影響。RV JR23株感染BHK21細胞6天后提取病毒RNA,利用上游引物C1和下游引物C2反轉錄擴增C基因,引物中分別含有EcoRⅠ和SacⅠ酶切位點,擴增的片斷酶切后,與經相同酶切的載體pBluescriptⅡSK~+片斷連接,測序證實成功構建重組載體pBSK-C。將pBSK-C單獨轉染至BHK21細胞中,間接免疫熒光(IFA)檢測表達蛋白活性,結果在核周區(qū)可見到較強的熒光。將pBSK-C與RV糖蛋白重組質粒pBSK-SPE2E1共同轉染BHK21細胞,Giemsa染色發(fā)現(xiàn)細胞幾乎全部發(fā)生融合,與單獨轉染pBSK-SPE2E1的細胞相比,細胞融合灶數(shù)量增多而且每個融合灶的細胞核數(shù)量增加,用指示基因法定量細胞融合顯示共轉染引起的細胞融合為糖蛋白單獨轉染的137%。可見CP可以促進RV包膜糖蛋白的細胞融合活性。 從本實驗結果可得出結論: RV包膜糖蛋白E1胞外功能區(qū)的10個二硫鍵都是維持E1細胞融合活性不可缺少的,其中C(5)-C(8)影響E1和E2的相互作用。 RV包膜糖蛋白E2序列中的14個半胱氨酸中,胞外區(qū)有1個半胱氨酸及胞質區(qū)的唯一1個半胱氨酸對E1的細胞融合活性沒有影響,其余12個都對E1的細胞融合功能有重要作用,它們可能通過二硫鍵的形成間接對其產生影響。 RV E1的213～285aa區(qū)域含有一些重要的細胞融合活性位點,是維持RV融合活性的關鍵氨基酸,如H238、P253、L257、D259G、D261、R266和P269。 成功構建了RV CP的重組載體,并在BHK21細胞中成功表達出CP,表達產物具有良好的生物學活性,能夠促進酸性條件下RV包膜糖蛋白的細胞融合活性。 本實驗為闡明RV引起細胞融合的分子機制、包膜糖蛋白的結構與功能研究奠定了堅實的基礎,也可以為RV致畸機制的研究提供幫助。
[Abstract]:The rubella virus (rubella virus, RV) is the only member of the family rash virus of the family apovirus. Human is the only natural host of RV,.RV natural infection only causes minor clinical symptoms. Many infections are asymptomatic subclinical infections. A rash occurs within 16~20 days of infection, first in the face, and then spread to the trunk and limbs, Other symptoms include low fever, enlarged lymph nodes, and sore throat. The most common complications of RV infection are arthritis and arthralgia, and most often occur in women.
The main problem caused by RV is its teratogenicity, the mother's pregnancy infection of RV can lead to a variety of clinical manifestations of the congenital rubella syndrome (CRS).CRS, the most common of which are deafness, including heart disease, mental retardation, and eye disorders such as cataracts and glaucoma. The earlier the infection of RV, the more serious the fetus is.
The mature RV virus particle is a spheroid with a diameter of 60NM. The core is the nucleocapsid of the capsid protein and the single strand positive chain genome 40S RNA, which is outsourced around the lipid bilayer membrane, the membrane is 5 to 6nm, which consists of glycoprotein E2 and E1. The capsid protein (capsid protein, CP) is a non glycosylated phosphate protein and forms the same by the two sulfur bond. Source two dimer.CP is rich in proline and arginine, and is related to the binding of RV genomic RNA.
Both E1 and E2 are type I membrane glycoproteins. The presence of.E1 and E2 in the form of different two polymers on the virus surface contains a segment of transmembrane region (TM), with a length of 22 and 39 amino acids. In E2, TM is a positive charge seven amino acid sequence and E1's 20 amino acid signal peptide.E1 and E2 are rich in cysteine residues and E1 extracellular All 20 cysteines in the functional region form a two sulfur bond, and E2 contains 14 cysteine residues.E1 with 3 N- glycosylation sites, and the number of N- glycosylation sites of E2 is different among different strains. In addition to N- glycosylation sites, E2 also contains O- linked glycosylation sites.
The function of E1 and E2 is widely studied. The study of monoclonal antibodies found that E1 contains at least 6 non repeated antigen sites, and that.E1 is related to the adsorption of hemagglutination and neutralization activity, which is also related to the adsorption of viruses and cells. It is difficult to study the function of the main surface protein.E2, because it is almost not exposed to the cell surface after it is combined with E1. Monoclonal antibodies could not be used to identify the antigenic sites. However, E2 also contained some hemagglutination epitopes and neutralization epitopes, as well as strain-specific epitopes.
Cell fusion is an important biological process for the invasion of cells including RV, including the invasion of cells, replication, release, transmission, and pathogenesis, and also an important step in the transmission of information between cells. The pathway of.RV invading cells is not clear, but there is evidence that E1 and E2 are found under the endocytic pathway into.Katow and Sugiura when pH is below 6. Conformational changes may occur, which is conducive to the fusion of viral envelope and inclusion membrane.
It is possible to eliminate or reduce the teratogenicity of RV by altering the mechanism of RV causing cell fusion and changing the genome of RV, changing its expression products and biological activity. It can also eliminate or reduce the risk of fetal infection by changing the characteristics of its intruding cells or replicating, and also to develop a more safe and effective new gene for RV. Engineering vaccine (gene deleted live vaccine, protein engineering vaccine, etc.) and specific antiviral polypeptide drugs laid the foundation.
Cell fusion is caused by protein on the surface of the cell, so the detection of cell fusion function requires that the protein expressed on the surface of the cell can express the signal peptide of.RV E2 on the processing and transport of the viral structural protein, in order to make the expressed protein properly processed and transshipped to the cell surface. In this study, the recombinant plasmid pBSK-SPE2E1 was constructed, and the sequence of signal peptide of E2, the whole gene sequence of E2 and E1 was cloned between the EcoR I and Xba I sites of the carrier pBluescript II SK~+, and a series of mutants were constructed by site directed and homologous recombination, and the cell melting was detected by Giemsa staining and indicator gene method. Activity changes, flow cytometry (FACS) detection of protein on the cell surface expression efficiency, Western blot detection of the total expression change, blood adsorption test to detect receptor recognition activity, analysis of mutation site on the RV envelope glycoprotein cell fusion activity, to determine the cell fusion activity site. This study also constructed the RV CP weight. The vector pBSK-C was used to detect the effect of CP on the fusion activity of the envelope glycoprotein.
1. The effect of two sulfur bonds in the extracellular domain of E1 on the fusion activity of RV cells
The extracellular function area of RV E1 envelope glycoprotein contains 20 cysteine residues and all forms intramolecular two sulfur bonds. In this study, 11 mutations of 20 cysteine in the extracellular domain of RV JR23 strain E1 protein were mutated into other amino acids by the combination of site directed and homologous recombination, and 11 mutant Cys2, Cys3, Cys4, Cys5, Cys6, were constructed. Cys8, Cys9, Cys12, Cys13, Cys17, and Cys20 were removed from each mutant to detect the effect of single disulfide bond deletion on the fusion activity of E1 cells.
Western blot results showed that the total expression of all the mutant proteins did not decrease, but the expression of Cys5 and Cys8 decreased sharply on the surface of the cells, so the two sulfur bond C (5) -C (8) could affect the interaction between E1 and E2, making them unable to form the hetero two polymer correctly, and thus could not be successfully transported to the cell surface. Mutant Cys2, C Ys6, Cys9, Cys12, Cys17 and Cys20 E1, the expression efficiency of E2 protein cells decreased compared with that of wild strains, so the two sulfur bonds formed by these cysteine may affect the interaction of E1, E2, and may also affect their intracellular transport. The expression efficiency of the mutant Cys3, Cys4 and Cys13, is 121% of the wild strain, respectively. Thus disulfide bonds formed by cysteine C(3), C(4) and C(13) have no effect on the interaction of envelope glycoproteins and their intracellular transport.
Although a large number of mutant proteins were expressed on the cell surface in most of the 11 mutants, no multinuclear giant cells were detected in all the transfected cells, so the 10 two sulfur bonds in the E1 extracellular domain were important for the cell fusion activity of RV.
Two, the effect of cysteine in E2 on the fusion activity of RV cells.
The RV envelope glycoprotein E2 contains 14 cysteines, of which 12 are located in the extracellular domain, 1 in the transmembrane region and 1 in the cytoplasm. In this study, 14 E2 cysteine mutants were constructed by site directed mutagenesis and homologous recombination in vivo, and one cysteine was removed by each mutant. The mutants were C69T, C82S, C91S, C124G, C132A, C139P, C152G, C157R, C172A, C196G, C207G, C196G, and, respectively.
Western blot showed that the changes in the 132nd and 219 cysteine residues in the 14 cysteine residues of E2 could lead to a decrease in the total expression of E1 protein, and the decrease of the cell fusion activity may be related to this. The changes in the remaining 12 cysteine did not affect the total expression of E1 protein, including C69T, C82S, C124G, C132A, C139P, C152G, C157R, C157R The 10 mutants of G, C219T and C255W almost lose cell fusion activity, indicating that in these mutants, E1 can not be transported to the cell surface effectively, or even though it can reach the cell surface, but the conformation changes can not be effectively exposed to the fusion active site.
The cell fusion activity of the mutant C91S and C259G is similar to that of the wild virus, indicating that the change of E2 ninety-first and 259th - cysteine does not affect the interaction of E1 and E2, so that E1 can be effectively transported to the cell surface to play its cell fusion.
The polyclonal antibody used in the experiment did not detect the mutant E2 protein. It was found that the cysteine residues of E2 had an important role in maintaining the conformation of the protein. The removal of cysteine directly led to the disappearance of antigenicity. Therefore, it is presumed that the cysteine of E2 may form a majority of the two sulfur bonds, indirectly affecting the cell fusion activity of RV.
Three, the effect of E1 key amino acid mutants on cell fusion activity.
Cysteine mutation analysis in the E1 extracellular domain showed that after 3,4 and 13 cysteine mutations, the cell surface expression was not decreased compared with the wild virus, but the fusion activity was not detected. The two sulfur bonds formed by the 3 cysteine were concentrated between the 213~285 amino acids of E1, and the region was rich in the RV neutralization epitopes. And hemagglutination inhibition epitopes, with more important biological activity, we selected some conservative or structural specific amino acids in this area, and constructed 12 mutants H226Q, H238Q, R252S, P253T, R254Q, R256T, L257T, D259G, D261G, P263A, R266Q, and P269S..
The mutant plasmids were transfected into BHK21 cells. After 24h, Giemsa staining was used to detect the cell fusion caused by the mutant proteins, and the mutant H226Q, R252S and R254Q were found to be similar to those of the wild strain, while the mutant R256T and P263A also could cause cell fusion, but the cell fusion intensity was lower than that of the wild strain. Mutants H238Q, P253T, L257T, D259G, D261G, R266Q and P269S only cause mild cell fusion and even fail to induce fusion.
Four, the effect of capsid protein CP on the fusion activity of RV envelope glycoprotein cells.
RV capsid protein CP plays a role in the process of viral replication, assembly and infection. The effect of this study on the cell fusion activity of the envelope glycoproteins was detected. The.RV JR23 strain infected BHK21 cells after 6 days to extract the virus RNA. The upstream primer C1 and the downstream primer C2 were used to amplify the C gene. The primers contained EcoR I and Sac I enzyme cut sites, and the amplification of the DNA was expanded. After the fragment of the fragment was cut, it was connected with the carrier pBluescript II SK~+, which was cut through the same enzyme. It was confirmed that the recombinant vector pBSK-C. was successfully constructed to transfect pBSK-C alone into BHK21 cells, and the activity of expression protein was detected by indirect immunofluorescence (IFA). The results showed that strong fluorescence could be seen in the peri nuclear region. PBSK-C and RV glycoprotein were recombinant plasmid pBSK-SPE2E1. BHK21 cells were transfected by CO transfection, and almost all the cells were fused by Giemsa staining. Compared with the cells transfected with pBSK-SPE2E1 alone, the number of cell fusion foci increased and the number of cell nuclei increased in each fusion focal point. The fusion of CO transfected cells by indicator gene quantitative cell fusion showed that the 137%. of the cells transfected by glycoprotein could be seen as C. P can promote the cell fusion activity of RV envelope glycoprotein.
The results can be concluded from the results of this experiment.
Ten disulfide bonds in the extracellular domain of RV envelope glycoprotein E1 are indispensable for maintaining the fusion activity of E1 cells, and C(5)-C(8) affects the interaction between E1 and E2.
In the 14 cysteine of RV envelope glycoprotein E2 sequence, the extracellular domain of 1 cysteine and the only 1 cysteine in the cytoplasmic region has no effect on the cell fusion activity of E1, and the other 12 are important for the cell fusion function of E1, and they may indirectly affect it through the formation of the two sulfur bond.
The 213 ~ 285aa region of RV E1 contains some important cell fusion active sites, which are key amino acids for the maintenance of RV fusion activity, such as H238, P253, L257, D259G, D261, R266 and P269..
The recombinant vector of RV CP was successfully constructed and CP was successfully expressed in BHK21 cells. The expression product has good biological activity, and it can promote the cell fusion activity of RV coated glycoprotein under acid condition.
This study provides a solid basis for elucidating the molecular mechanism of RV induced cell fusion, the structure and function of the membrane glycoproteins, and can also help the research of the RV teratogenicity mechanism.
【學位授予單位】：山東大學
【學位級別】：博士
【學位授予年份】：2009
【分類號】：R373
本文編號：2167302
[Abstract]:The rubella virus (rubella virus, RV) is the only member of the family rash virus of the family apovirus. Human is the only natural host of RV,.RV natural infection only causes minor clinical symptoms. Many infections are asymptomatic subclinical infections. A rash occurs within 16~20 days of infection, first in the face, and then spread to the trunk and limbs, Other symptoms include low fever, enlarged lymph nodes, and sore throat. The most common complications of RV infection are arthritis and arthralgia, and most often occur in women.
The main problem caused by RV is its teratogenicity, the mother's pregnancy infection of RV can lead to a variety of clinical manifestations of the congenital rubella syndrome (CRS).CRS, the most common of which are deafness, including heart disease, mental retardation, and eye disorders such as cataracts and glaucoma. The earlier the infection of RV, the more serious the fetus is.
The mature RV virus particle is a spheroid with a diameter of 60NM. The core is the nucleocapsid of the capsid protein and the single strand positive chain genome 40S RNA, which is outsourced around the lipid bilayer membrane, the membrane is 5 to 6nm, which consists of glycoprotein E2 and E1. The capsid protein (capsid protein, CP) is a non glycosylated phosphate protein and forms the same by the two sulfur bond. Source two dimer.CP is rich in proline and arginine, and is related to the binding of RV genomic RNA.
Both E1 and E2 are type I membrane glycoproteins. The presence of.E1 and E2 in the form of different two polymers on the virus surface contains a segment of transmembrane region (TM), with a length of 22 and 39 amino acids. In E2, TM is a positive charge seven amino acid sequence and E1's 20 amino acid signal peptide.E1 and E2 are rich in cysteine residues and E1 extracellular All 20 cysteines in the functional region form a two sulfur bond, and E2 contains 14 cysteine residues.E1 with 3 N- glycosylation sites, and the number of N- glycosylation sites of E2 is different among different strains. In addition to N- glycosylation sites, E2 also contains O- linked glycosylation sites.
The function of E1 and E2 is widely studied. The study of monoclonal antibodies found that E1 contains at least 6 non repeated antigen sites, and that.E1 is related to the adsorption of hemagglutination and neutralization activity, which is also related to the adsorption of viruses and cells. It is difficult to study the function of the main surface protein.E2, because it is almost not exposed to the cell surface after it is combined with E1. Monoclonal antibodies could not be used to identify the antigenic sites. However, E2 also contained some hemagglutination epitopes and neutralization epitopes, as well as strain-specific epitopes.
Cell fusion is an important biological process for the invasion of cells including RV, including the invasion of cells, replication, release, transmission, and pathogenesis, and also an important step in the transmission of information between cells. The pathway of.RV invading cells is not clear, but there is evidence that E1 and E2 are found under the endocytic pathway into.Katow and Sugiura when pH is below 6. Conformational changes may occur, which is conducive to the fusion of viral envelope and inclusion membrane.
It is possible to eliminate or reduce the teratogenicity of RV by altering the mechanism of RV causing cell fusion and changing the genome of RV, changing its expression products and biological activity. It can also eliminate or reduce the risk of fetal infection by changing the characteristics of its intruding cells or replicating, and also to develop a more safe and effective new gene for RV. Engineering vaccine (gene deleted live vaccine, protein engineering vaccine, etc.) and specific antiviral polypeptide drugs laid the foundation.
Cell fusion is caused by protein on the surface of the cell, so the detection of cell fusion function requires that the protein expressed on the surface of the cell can express the signal peptide of.RV E2 on the processing and transport of the viral structural protein, in order to make the expressed protein properly processed and transshipped to the cell surface. In this study, the recombinant plasmid pBSK-SPE2E1 was constructed, and the sequence of signal peptide of E2, the whole gene sequence of E2 and E1 was cloned between the EcoR I and Xba I sites of the carrier pBluescript II SK~+, and a series of mutants were constructed by site directed and homologous recombination, and the cell melting was detected by Giemsa staining and indicator gene method. Activity changes, flow cytometry (FACS) detection of protein on the cell surface expression efficiency, Western blot detection of the total expression change, blood adsorption test to detect receptor recognition activity, analysis of mutation site on the RV envelope glycoprotein cell fusion activity, to determine the cell fusion activity site. This study also constructed the RV CP weight. The vector pBSK-C was used to detect the effect of CP on the fusion activity of the envelope glycoprotein.
1. The effect of two sulfur bonds in the extracellular domain of E1 on the fusion activity of RV cells
The extracellular function area of RV E1 envelope glycoprotein contains 20 cysteine residues and all forms intramolecular two sulfur bonds. In this study, 11 mutations of 20 cysteine in the extracellular domain of RV JR23 strain E1 protein were mutated into other amino acids by the combination of site directed and homologous recombination, and 11 mutant Cys2, Cys3, Cys4, Cys5, Cys6, were constructed. Cys8, Cys9, Cys12, Cys13, Cys17, and Cys20 were removed from each mutant to detect the effect of single disulfide bond deletion on the fusion activity of E1 cells.
Western blot results showed that the total expression of all the mutant proteins did not decrease, but the expression of Cys5 and Cys8 decreased sharply on the surface of the cells, so the two sulfur bond C (5) -C (8) could affect the interaction between E1 and E2, making them unable to form the hetero two polymer correctly, and thus could not be successfully transported to the cell surface. Mutant Cys2, C Ys6, Cys9, Cys12, Cys17 and Cys20 E1, the expression efficiency of E2 protein cells decreased compared with that of wild strains, so the two sulfur bonds formed by these cysteine may affect the interaction of E1, E2, and may also affect their intracellular transport. The expression efficiency of the mutant Cys3, Cys4 and Cys13, is 121% of the wild strain, respectively. Thus disulfide bonds formed by cysteine C(3), C(4) and C(13) have no effect on the interaction of envelope glycoproteins and their intracellular transport.
Although a large number of mutant proteins were expressed on the cell surface in most of the 11 mutants, no multinuclear giant cells were detected in all the transfected cells, so the 10 two sulfur bonds in the E1 extracellular domain were important for the cell fusion activity of RV.
Two, the effect of cysteine in E2 on the fusion activity of RV cells.
The RV envelope glycoprotein E2 contains 14 cysteines, of which 12 are located in the extracellular domain, 1 in the transmembrane region and 1 in the cytoplasm. In this study, 14 E2 cysteine mutants were constructed by site directed mutagenesis and homologous recombination in vivo, and one cysteine was removed by each mutant. The mutants were C69T, C82S, C91S, C124G, C132A, C139P, C152G, C157R, C172A, C196G, C207G, C196G, and, respectively.
Western blot showed that the changes in the 132nd and 219 cysteine residues in the 14 cysteine residues of E2 could lead to a decrease in the total expression of E1 protein, and the decrease of the cell fusion activity may be related to this. The changes in the remaining 12 cysteine did not affect the total expression of E1 protein, including C69T, C82S, C124G, C132A, C139P, C152G, C157R, C157R The 10 mutants of G, C219T and C255W almost lose cell fusion activity, indicating that in these mutants, E1 can not be transported to the cell surface effectively, or even though it can reach the cell surface, but the conformation changes can not be effectively exposed to the fusion active site.
The cell fusion activity of the mutant C91S and C259G is similar to that of the wild virus, indicating that the change of E2 ninety-first and 259th - cysteine does not affect the interaction of E1 and E2, so that E1 can be effectively transported to the cell surface to play its cell fusion.
The polyclonal antibody used in the experiment did not detect the mutant E2 protein. It was found that the cysteine residues of E2 had an important role in maintaining the conformation of the protein. The removal of cysteine directly led to the disappearance of antigenicity. Therefore, it is presumed that the cysteine of E2 may form a majority of the two sulfur bonds, indirectly affecting the cell fusion activity of RV.
Three, the effect of E1 key amino acid mutants on cell fusion activity.
Cysteine mutation analysis in the E1 extracellular domain showed that after 3,4 and 13 cysteine mutations, the cell surface expression was not decreased compared with the wild virus, but the fusion activity was not detected. The two sulfur bonds formed by the 3 cysteine were concentrated between the 213~285 amino acids of E1, and the region was rich in the RV neutralization epitopes. And hemagglutination inhibition epitopes, with more important biological activity, we selected some conservative or structural specific amino acids in this area, and constructed 12 mutants H226Q, H238Q, R252S, P253T, R254Q, R256T, L257T, D259G, D261G, P263A, R266Q, and P269S..
The mutant plasmids were transfected into BHK21 cells. After 24h, Giemsa staining was used to detect the cell fusion caused by the mutant proteins, and the mutant H226Q, R252S and R254Q were found to be similar to those of the wild strain, while the mutant R256T and P263A also could cause cell fusion, but the cell fusion intensity was lower than that of the wild strain. Mutants H238Q, P253T, L257T, D259G, D261G, R266Q and P269S only cause mild cell fusion and even fail to induce fusion.
Four, the effect of capsid protein CP on the fusion activity of RV envelope glycoprotein cells.
RV capsid protein CP plays a role in the process of viral replication, assembly and infection. The effect of this study on the cell fusion activity of the envelope glycoproteins was detected. The.RV JR23 strain infected BHK21 cells after 6 days to extract the virus RNA. The upstream primer C1 and the downstream primer C2 were used to amplify the C gene. The primers contained EcoR I and Sac I enzyme cut sites, and the amplification of the DNA was expanded. After the fragment of the fragment was cut, it was connected with the carrier pBluescript II SK~+, which was cut through the same enzyme. It was confirmed that the recombinant vector pBSK-C. was successfully constructed to transfect pBSK-C alone into BHK21 cells, and the activity of expression protein was detected by indirect immunofluorescence (IFA). The results showed that strong fluorescence could be seen in the peri nuclear region. PBSK-C and RV glycoprotein were recombinant plasmid pBSK-SPE2E1. BHK21 cells were transfected by CO transfection, and almost all the cells were fused by Giemsa staining. Compared with the cells transfected with pBSK-SPE2E1 alone, the number of cell fusion foci increased and the number of cell nuclei increased in each fusion focal point. The fusion of CO transfected cells by indicator gene quantitative cell fusion showed that the 137%. of the cells transfected by glycoprotein could be seen as C. P can promote the cell fusion activity of RV envelope glycoprotein.
The results can be concluded from the results of this experiment.
Ten disulfide bonds in the extracellular domain of RV envelope glycoprotein E1 are indispensable for maintaining the fusion activity of E1 cells, and C(5)-C(8) affects the interaction between E1 and E2.
In the 14 cysteine of RV envelope glycoprotein E2 sequence, the extracellular domain of 1 cysteine and the only 1 cysteine in the cytoplasmic region has no effect on the cell fusion activity of E1, and the other 12 are important for the cell fusion function of E1, and they may indirectly affect it through the formation of the two sulfur bond.
The 213 ~ 285aa region of RV E1 contains some important cell fusion active sites, which are key amino acids for the maintenance of RV fusion activity, such as H238, P253, L257, D259G, D261, R266 and P269..
The recombinant vector of RV CP was successfully constructed and CP was successfully expressed in BHK21 cells. The expression product has good biological activity, and it can promote the cell fusion activity of RV coated glycoprotein under acid condition.
This study provides a solid basis for elucidating the molecular mechanism of RV induced cell fusion, the structure and function of the membrane glycoproteins, and can also help the research of the RV teratogenicity mechanism.
【學位授予單位】：山東大學
【學位級別】：博士
【學位授予年份】：2009
【分類號】：R373
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