人類腸道病毒71型衣殼蛋白全基因成和原核表達分析
發(fā)布時間：2019-01-16 04:23
【摘要】：人類腸道病毒71型（Human enterovirus71，簡稱EV71）屬于小RNA病毒科腸道病毒屬，人類腸道病毒A，核酸為單股正鏈RNA。目前已知EV71的感染可以導致手足口病、無菌性腦膜炎、腦炎、皰疹性咽峽炎和脊髓灰質(zhì)炎樣的麻痹性疾病等多種與神經(jīng)系統(tǒng)相關的疾病。手足口病在美國和歐洲處于小范圍流行的階段，但是在1975保加利亞和1978年匈牙利有過兩次大疫情爆發(fā)和高死亡率。近年來，EV71病毒的流行在亞太地區(qū)呈上升趨勢，包括馬來西亞、新加坡、臺灣、日本、韓國、越南和中國大陸。 腸道病毒EV71型有A、B（B1、B2、B3、B4、B5）、C（C1、C2、C3、C4、C5）三個基因型，共11個亞型，病毒的顆粒為二十面體立體對稱的球形結構，無包膜，直徑約24～30nm，核酸為含有大約7400個核苷酸的單股正鏈RNA。RNA中僅有一個開放閱讀框（ORF），編碼含2194個氨基酸的多聚蛋白，在其兩側為5′和3′非編碼區(qū)（UTRs），與其他腸道RNA病毒一樣，在3′末端有多聚腺苷酸（polyA）尾，而其5′末端共價結合有一個小分子量的蛋白（VPg）VP1，P2和VP3三個蛋白暴露在病毒外殼的表面，而VP4包埋在病毒外殼的內(nèi)側與病毒核心緊密連接，因而抗原決定簇基本上位于VP1-VP3上。 近年來，已經(jīng)開發(fā)出不同類型的EV71疫苗，但目前普遍處于臨床前開發(fā)階段，包括滅活疫苗、DNA疫苗、減毒活疫苗、亞單位疫苗、表位多肽疫苗和病毒樣顆粒疫苗。這些疫苗在動物模型中的研究表明中和抗體的產(chǎn)生起關鍵作用。本研究就目前研究中缺乏批準上市的抗原、抗體（酶標或化學發(fā)光）試劑，造成疫苗活性評價中中和抗體效價測定的不準確性和可比性，我們構建了VP1，VP0，VP3三種原核表達質(zhì)粒，成功的表達了VP1，VP0蛋白，并對其免疫原性做了初步評價而且驗證了VP1是抗原決定中心，為EV71診斷試劑盒和亞單位疫苗的研究奠定基礎。 （一） EV71相關基因片段的體外合成 根據(jù)GenBank中錄入的EV71/Fuyang.Anhui.CHN/19.08/6株全基因序列，翻譯成經(jīng)大腸桿菌密碼子優(yōu)化后的VP1，VP0,VP3的基因核苷酸序列，利用OE-PCR原理設計全基因體外合成的特異性引物并進行體外合成。合成的全長片段TA克隆至pMD18T載體上，經(jīng)過序列測定和最后的修正合成，成功合成三個外殼蛋白的基因片段。 （二）VP1，VP0，VP3的原核表達和VP1的免疫原性分析。 分別以三個測序正確的含有基因片段的質(zhì)粒為模板，用含酶切位點的引物PCR擴增出DNA產(chǎn)物后克隆到原核表達載體pET-32a，分別構建的三種原核表達質(zhì)粒pET-32a-VP1，pET-32a-VP0，pET-32a-VP3將三種原核表達質(zhì)粒轉入E.coliBL21(DE3)，經(jīng)過IPTG誘導表達和純化，，進行動物免疫EV71-VP1蛋白的動物免疫，對其免疫原性進行分析。成功構建了三個原核表達質(zhì)粒對進行原核表達并用Ni-NTA親和層析法純化目的融合蛋白。其中VP1，VP0表達量較高，VP3表達量不高。VP1免疫新西蘭大白兔和BABL/C小鼠并制備抗血清。ELISA檢測pET-32a-VP1和手足口病患兒的血清呈特異性結合反應，其IgG抗體滴度達1：64000，pET-32a-VP0和手足口病抗體滴度:1：32000，pET32a蛋白與僅有患兒的血清較弱結合。說明我們利用原核密碼子體外基因合成表達表達純化的蛋白很好的保留了很好的抗原性，同時也反映了VP1是抗原決定中心。為今后EV71診斷試劑盒和亞單位疫苗的研究提供參考。 （三）基孔肯雅病毒衣殼蛋白C和包膜蛋白E2的全基因合成和原核表達分析 利用重疊延伸PCR方法體外合成基孔肯雅病毒外殼蛋白C和包膜蛋白E2的全基因序列，并且構建外殼蛋白C和包膜蛋白E2的原核表達質(zhì)粒。同時根據(jù)Expasy軟件對E2跨膜疏水的預測，構建缺失疏水區(qū)的E2(1-350)突變體原核表達質(zhì)粒。將測序正確的原核質(zhì)粒轉化至BL21(DE3)，經(jīng)IPTG誘導表達，SDS-PAGE檢測重組質(zhì)粒的表達情況。經(jīng)IPTG誘導表達后，SDS-PAGE結果顯示缺失突變體pET21b-E2(1-350)融合蛋白表達量較pET21b-E2(1-404)有明顯提高。提示E2蛋白跨膜疏水區(qū)（351-378aa）對該蛋白的原核表達具有重要影響。
[Abstract]:Human enterovirus 71 (EV71) belongs to the genus Enterovirus of the small RNA viridae, and the human enterovirus A and the nucleic acid are single-stranded positive-chain RNA. At present, it is known that the infection of the EV71 can lead to a variety of nervous system-related diseases such as hand-foot-mouth disease, aseptic meningitis, encephalitis, herpetic angina, and poliomyelitis-like paralysis. Hand-foot-mouth disease is in a small-scale epidemic in the United States and Europe, but in 1975 and 1978, there were two large outbreaks and high mortality rates in Hungary. In recent years, the prevalence of the EV71 virus is on the rise in the Asia-Pacific region, including Malaysia, Singapore, Taiwan, Japan, South Korea, Vietnam and China. The EV71 of the enterovirus type is A, B (B1, B2, B3, B4, B5), C (C1, C2, C3, C4, C5) three genotypes, 11 subtypes, the particle of the virus is the spherical structure of the icosahedral three-dimensional symmetry, the non-enveloped, the diameter is about 24 to 30n. m, the nucleic acid is a single-stranded positive strand RNA containing about 7400 nucleotides. There is only one open reading frame (ORF) in the RNA, which encodes a multimeric protein containing 2194 amino acids, with both 5 and 3 non-coding regions (UTRs) on both sides thereof, and one of the other intestinal RNA viruses. For example, polyadenosanoic acid (polyA) tail is present at the end of 3, and the 5-terminal end of which is covalently bound to a small molecular weight protein (VPg) VP1, P2 and VP3 three proteins are exposed to the surface of the viral envelope, while VP4 is embedded in the inner side of the viral envelope and is in close contact with the virus core and thus the antigenic determinant is located substantially in the VP1-VP3 In recent years, different types of EV71 vaccines have been developed, but are currently in the pre-clinical development phase, including live vaccines, DNA vaccines, attenuated live vaccines, subunit vaccines, epitope polypeptide vaccines, and virus-like The study of these vaccines in animal models shows that the production of neutralizing antibodies is off The key effect of this study is that the present study lacks the reagent of the antigen, antibody (enzyme or chemiluminescence) to be listed in the present study, which results in the inaccuracy and comparability of the antibody titer determination in the evaluation of the vaccine activity. We construct three prokaryotic expression plasmids of VP1, VP0 and VP3, which are successfully expressed. VP1, VP0 protein, and the immunogenicity of VP1 and VP0 were first evaluated, and VP1 was the antigen-determining center, and it was the study of the EV71 diagnostic kit and subunit vaccine. Base. (I) EV71-related gene chip The in vitro synthesis of the segment is based on the whole gene sequence of the EV71/ Fuyang, Anhua. CHN/ 19. 08/ 6 strain entered in the GenBank, and is translated into the gene core acid sequence of VP1, VP0 and VP3 after being optimized by the E. coli codon, and the specific primer for the in vitro synthesis of the whole gene is designed by the OE-PCR principle. The synthetic full-length fragment TA is cloned into the pMD18T vector, and the three shells are successfully synthesized through the sequence measurement and the final modified synthesis. Prokaryotic Expression and V of VP1, VPO, VP3 and the three prokaryotic expression plasmids pET-32a-VP1, pET-32a-VP0 and pET-32a-VP3 were respectively constructed to transfer three prokaryotic expression plasmids to E. co. coli BL21 (DE3), and expressed and purified by IPTG, and the animal immunity of the animal immune EV71-VP1 protein is carried out and the immunogenicity of the three prokaryotic expression plasmids is analyzed, and the three prokaryotic expression plasmids are successfully constructed for prokaryotic expression and are affinity with the Ni-NTA. The target fusion protein is purified by a chromatography method, wherein the expression amount of VP1 and VP0 High, VP3 expression is not high. VP1 immune New Zealand white rabbits and BAB The serum of pET-32a-VP1 and hand-foot-mouth disease was detected by ELISA. The concentration of IgG antibody was 1: 64000, the level of pET-32a-VPO and the degree of antibody drop of hand-foot-mouth disease were 1: 32000 and the protein of pET32a. Only the serum of the children is weak binding. The expression of the purified protein by using the in vitro gene synthesis of the prokaryote is very good, the antigenicity is good, VP1 is an antigen-determining center. For future EV71 diagnostic kits and subs Reference to the study of the unit vaccine. (iii) the base-hole Kenya virus capsid protein C and the envelope protein E 2. The whole-gene synthesis and the prokaryotic expression analysis use the overlapping extension PCR method to synthesize the shell egg of the kaphenya virus in vitro. The whole gene sequence of the white C and the envelope protein E2, and the shell protein is constructed the prokaryotic expression plasmid of the C and the envelope protein E2 is constructed, and the E2 (E2 (E2) of the missing hydrophobic region is constructed according to the prediction of the E2 cross-membrane hydrophobic according to the expression of the Expression software. 1-350) the prokaryotic expression plasmid of the mutant. The correct prokaryotic plasmid is transformed into the BL21 (DE3), and the expression is induced by IPTG, and the SDS-The expression of the recombinant plasmid was detected by PAGE. After the expression of IPTG, the expression of the fusion protein of the deleted mutant pET21b-E2 (1-350) was higher than that of the pET21b.-E2 (1-404) was significantly improved. It was suggested that E2 protein cross-membrane hydrophobic region (351-378aa)
【學位授予單位】：安徽醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R373
本文編號：2409458
[Abstract]:Human enterovirus 71 (EV71) belongs to the genus Enterovirus of the small RNA viridae, and the human enterovirus A and the nucleic acid are single-stranded positive-chain RNA. At present, it is known that the infection of the EV71 can lead to a variety of nervous system-related diseases such as hand-foot-mouth disease, aseptic meningitis, encephalitis, herpetic angina, and poliomyelitis-like paralysis. Hand-foot-mouth disease is in a small-scale epidemic in the United States and Europe, but in 1975 and 1978, there were two large outbreaks and high mortality rates in Hungary. In recent years, the prevalence of the EV71 virus is on the rise in the Asia-Pacific region, including Malaysia, Singapore, Taiwan, Japan, South Korea, Vietnam and China. The EV71 of the enterovirus type is A, B (B1, B2, B3, B4, B5), C (C1, C2, C3, C4, C5) three genotypes, 11 subtypes, the particle of the virus is the spherical structure of the icosahedral three-dimensional symmetry, the non-enveloped, the diameter is about 24 to 30n. m, the nucleic acid is a single-stranded positive strand RNA containing about 7400 nucleotides. There is only one open reading frame (ORF) in the RNA, which encodes a multimeric protein containing 2194 amino acids, with both 5 and 3 non-coding regions (UTRs) on both sides thereof, and one of the other intestinal RNA viruses. For example, polyadenosanoic acid (polyA) tail is present at the end of 3, and the 5-terminal end of which is covalently bound to a small molecular weight protein (VPg) VP1, P2 and VP3 three proteins are exposed to the surface of the viral envelope, while VP4 is embedded in the inner side of the viral envelope and is in close contact with the virus core and thus the antigenic determinant is located substantially in the VP1-VP3 In recent years, different types of EV71 vaccines have been developed, but are currently in the pre-clinical development phase, including live vaccines, DNA vaccines, attenuated live vaccines, subunit vaccines, epitope polypeptide vaccines, and virus-like The study of these vaccines in animal models shows that the production of neutralizing antibodies is off The key effect of this study is that the present study lacks the reagent of the antigen, antibody (enzyme or chemiluminescence) to be listed in the present study, which results in the inaccuracy and comparability of the antibody titer determination in the evaluation of the vaccine activity. We construct three prokaryotic expression plasmids of VP1, VP0 and VP3, which are successfully expressed. VP1, VP0 protein, and the immunogenicity of VP1 and VP0 were first evaluated, and VP1 was the antigen-determining center, and it was the study of the EV71 diagnostic kit and subunit vaccine. Base. (I) EV71-related gene chip The in vitro synthesis of the segment is based on the whole gene sequence of the EV71/ Fuyang, Anhua. CHN/ 19. 08/ 6 strain entered in the GenBank, and is translated into the gene core acid sequence of VP1, VP0 and VP3 after being optimized by the E. coli codon, and the specific primer for the in vitro synthesis of the whole gene is designed by the OE-PCR principle. The synthetic full-length fragment TA is cloned into the pMD18T vector, and the three shells are successfully synthesized through the sequence measurement and the final modified synthesis. Prokaryotic Expression and V of VP1, VPO, VP3 and the three prokaryotic expression plasmids pET-32a-VP1, pET-32a-VP0 and pET-32a-VP3 were respectively constructed to transfer three prokaryotic expression plasmids to E. co. coli BL21 (DE3), and expressed and purified by IPTG, and the animal immunity of the animal immune EV71-VP1 protein is carried out and the immunogenicity of the three prokaryotic expression plasmids is analyzed, and the three prokaryotic expression plasmids are successfully constructed for prokaryotic expression and are affinity with the Ni-NTA. The target fusion protein is purified by a chromatography method, wherein the expression amount of VP1 and VP0 High, VP3 expression is not high. VP1 immune New Zealand white rabbits and BAB The serum of pET-32a-VP1 and hand-foot-mouth disease was detected by ELISA. The concentration of IgG antibody was 1: 64000, the level of pET-32a-VPO and the degree of antibody drop of hand-foot-mouth disease were 1: 32000 and the protein of pET32a. Only the serum of the children is weak binding. The expression of the purified protein by using the in vitro gene synthesis of the prokaryote is very good, the antigenicity is good, VP1 is an antigen-determining center. For future EV71 diagnostic kits and subs Reference to the study of the unit vaccine. (iii) the base-hole Kenya virus capsid protein C and the envelope protein E 2. The whole-gene synthesis and the prokaryotic expression analysis use the overlapping extension PCR method to synthesize the shell egg of the kaphenya virus in vitro. The whole gene sequence of the white C and the envelope protein E2, and the shell protein is constructed the prokaryotic expression plasmid of the C and the envelope protein E2 is constructed, and the E2 (E2 (E2) of the missing hydrophobic region is constructed according to the prediction of the E2 cross-membrane hydrophobic according to the expression of the Expression software. 1-350) the prokaryotic expression plasmid of the mutant. The correct prokaryotic plasmid is transformed into the BL21 (DE3), and the expression is induced by IPTG, and the SDS-The expression of the recombinant plasmid was detected by PAGE. After the expression of IPTG, the expression of the fusion protein of the deleted mutant pET21b-E2 (1-350) was higher than that of the pET21b.-E2 (1-404) was significantly improved. It was suggested that E2 protein cross-membrane hydrophobic region (351-378aa)
【學位授予單位】：安徽醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R373
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本文編號：2409458
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