【摘要】： 日本血吸蟲(chóng)病(schistosomiasis japonica)作為人獸共患寄生蟲(chóng)病,在我國(guó)仍然是一個(gè)重要的公共衛(wèi)生問(wèn)題�，F(xiàn)階段我國(guó)實(shí)行的是“以控制傳染源”為主、綜合治理的血吸蟲(chóng)病防治策略。其中,吡喹酮群體化療是目前血吸蟲(chóng)病防治措施的基礎(chǔ),但血吸蟲(chóng)病仍在向新的地區(qū)蔓延,并且吡喹酮化療也有一定的局限性,群體化療并不能防止重復(fù)感染,還可能會(huì)產(chǎn)生吡喹酮抗性株。因此,在血吸蟲(chóng)病防治策略中,疫苗策略已被認(rèn)為將是吡喹酮化療措施的重要補(bǔ)充。目前已有酶性、肌性、膜相關(guān)性等多種疫苗候選抗原的基因得到了克隆和表達(dá),并且開(kāi)展了動(dòng)物保護(hù)性實(shí)驗(yàn),雖然取得了一定的預(yù)期效果,但繼續(xù)尋找新的候選抗原分子以及提高候選疫苗分子的免疫原性仍是血吸蟲(chóng)病疫苗研究的重要方向,通過(guò)免疫篩選cDNA文庫(kù)去發(fā)掘更有效的疫苗候選分子是獲得新的候選抗原分子的有效途徑。 發(fā)展疫苗的主要目的是降低蟲(chóng)荷和蟲(chóng)卵在肝組織的沉積,因此疫苗的效應(yīng)應(yīng)多針對(duì)童蟲(chóng)階段和成蟲(chóng)產(chǎn)卵。東方田鼠(Microtus fortis,Mf)是一種感染日本血吸蟲(chóng)后不致病的哺乳類動(dòng)物。血吸蟲(chóng)童蟲(chóng)可能是宿主免疫系統(tǒng)較為適合的靶子,若能找到童蟲(chóng)階段的特異性抗原分子,將血吸蟲(chóng)消滅在此階段或阻止其生長(zhǎng)、發(fā)育、成熟、產(chǎn)卵和致病,這不僅可以減輕血吸蟲(chóng)病所造成的病理?yè)p害,還可有效地阻止其傳播。因此用東方田鼠血清免疫篩選日本血吸蟲(chóng)童蟲(chóng)cDNA文庫(kù)中,以尋找相關(guān)的疫苗候選分子,可能會(huì)獲得令人滿意的結(jié)果,并為東方田鼠天然抗日本血吸蟲(chóng)機(jī)制的研究提供信息。 精氨酸甲基化在血吸蟲(chóng)基因表達(dá)調(diào)節(jié)中起著重要的作用。這是一種翻譯后修飾,它參與了多種細(xì)胞功能,包括RNA加工處理、細(xì)胞信號(hào)轉(zhuǎn)導(dǎo)、蛋白亞細(xì)胞定位、轉(zhuǎn)錄后調(diào)節(jié)和DNA修復(fù)。 高遷移率族蛋白B1(HMGB1)參與基因轉(zhuǎn)錄、復(fù)制、重組與修復(fù)。胞外HMGB1是一種重要的晚期炎癥介質(zhì),它可以激活巨噬細(xì)胞釋放TNF-α和IL-13等早期炎癥因子。在血吸蟲(chóng)感染中,TNF-α和IL-13對(duì)蟲(chóng)卵周圍肉芽腫的形成起著重要的免疫誘導(dǎo)作用,可能是宿主感染后免疫調(diào)節(jié)的關(guān)鍵分子。HMGB1與一些感染性疾病的發(fā)病密切相關(guān)。 本研究從日本血吸蟲(chóng)童蟲(chóng)cDNA文庫(kù)篩選得到的陽(yáng)性克隆中選擇了蛋白質(zhì)精氨酸甲基轉(zhuǎn)移酶1(PRMT1)和高遷移率族蛋白B1(HMGB1)編碼基因進(jìn)行研究。首先通過(guò)生物信息學(xué)分析獲得其完整的開(kāi)放閱讀框,然后通過(guò)分子克隆技術(shù)對(duì)這2個(gè)基因進(jìn)行了克隆表達(dá)。隨后,對(duì)純化重組蛋白reSjcHMGB1開(kāi)展了動(dòng)物免疫保護(hù)性試驗(yàn),評(píng)價(jià)其作為疫苗候選抗原的價(jià)值。 一、東方田鼠日本血吸蟲(chóng)天然抗性相關(guān)基因的免疫篩選 用日本血吸蟲(chóng)天然抗性東方田鼠血清免疫篩選日本血吸蟲(chóng)肝期童蟲(chóng)cDNA文庫(kù),將3次復(fù)篩獲得的陽(yáng)性克隆轉(zhuǎn)入大腸桿菌(E.coil)BM25.8環(huán)化成質(zhì)粒,抽提質(zhì)粒DNA,EcoRⅠ和HindⅢ雙酶切瓊脂糖凝膠電泳鑒定,插入片段進(jìn)行核苷酸序列測(cè)序,并進(jìn)行生物信息學(xué)分析。結(jié)果,經(jīng)3次復(fù)篩后獲得32個(gè)陽(yáng)性克隆,插入片段為300 bp～1 100 bp之間,測(cè)序結(jié)果經(jīng)同源性分析,共獲得26個(gè)不同分子基因:高遷移率族蛋白B1(HMGB1)部分基因,蛋白質(zhì)精氨酸甲基轉(zhuǎn)移酶部分編碼基因,細(xì)胞色素b部分編碼基因,線粒體編碼區(qū)基因,16個(gè)日本血吸蟲(chóng)未知蛋白編碼基因,6個(gè)日本血吸蟲(chóng)未知新基因。本研究用東方田鼠血清篩選日本血吸蟲(chóng)童蟲(chóng)cDNA文庫(kù),獲得一批新的日本血吸蟲(chóng)疫苗候選分子的編碼基因,為研究血吸蟲(chóng)病疫苗和血吸蟲(chóng)病免疫診斷奠定了基礎(chǔ)。 二、日本血吸蟲(chóng)蛋白質(zhì)精氨酸甲基轉(zhuǎn)移酶(PRMT)1編碼基因的克隆、表達(dá)和分析 依據(jù)電子延伸得到的SjPRMT1基因序列設(shè)計(jì)一對(duì)引物,上游引物引入BamHⅠ酶切位點(diǎn),下游引物引入XhoⅠ酶切位點(diǎn)。以日本血吸蟲(chóng)成蟲(chóng)總RNA為模板,經(jīng)反轉(zhuǎn)錄PCR(RT-PCR)擴(kuò)增目的編碼基因。純化PCR產(chǎn)物與pGEM-T載體連接后轉(zhuǎn)化感受態(tài)大腸桿菌JM109,抽提重組質(zhì)粒DNA用BamHⅠ和XhoⅠ雙酶切及核苷酸序列測(cè)序進(jìn)行鑒定。選擇閱讀框正確的克隆,純化重組質(zhì)粒中目的基因雙酶切片段,亞克隆入pET28a原核表達(dá)載體,構(gòu)建重組質(zhì)粒pET28a-SjPRMT1,轉(zhuǎn)化DH5α感受態(tài)菌,重組質(zhì)粒經(jīng)雙酶切和核苷酸序列鑒定后,陽(yáng)性克隆質(zhì)粒轉(zhuǎn)化感受態(tài)大腸桿菌BL21(DE3),IPTG誘導(dǎo)表達(dá)并獲得純化的重組蛋白(簡(jiǎn)稱為reSjPRMT1),采用SDS-PAGE和Western blotting分析和鑒定該重組蛋白。運(yùn)用Gene Runner軟件預(yù)測(cè)reSjPRMT1蛋白的二級(jí)結(jié)構(gòu)、功能位點(diǎn)及表位特征。結(jié)果,RT-PCR擴(kuò)增出一大小與預(yù)期一致的基因片段。TA克隆插入目的片段經(jīng)核苷酸序列測(cè)定,cDNA全長(zhǎng)1083 bp,編碼360個(gè)氨基酸。序列分析表明該片段與SmPRMT1基因序列同源性為87%,推導(dǎo)的氨基酸序列同源性為95%。表達(dá)蛋白經(jīng)SDS-PAGE和Western blotting分析顯示,reSjPRMT1重組蛋白的分子質(zhì)量約43 kDa(包括6個(gè)組氨酸),以可溶性方式表達(dá),可被日本血吸蟲(chóng)感染小鼠血清和抗His-G HRP抗體識(shí)別。SjPRMT1基因的克隆、表達(dá)獲得成功,并獲得純化的重組蛋白,為今后進(jìn)一步研究其生物學(xué)特性以及免疫原性奠定了基礎(chǔ)。 三、日本血吸蟲(chóng)高遷移率族蛋白B1(HMGB1)編碼基因的克隆、表達(dá)和免疫保護(hù)性研究 依據(jù)公布的SmHMGB1基因序列設(shè)計(jì)一對(duì)簡(jiǎn)并引物,上游引物引入BamHⅠ酶切位點(diǎn),下游引物引入SalⅠ酶切位點(diǎn)。以日本血吸蟲(chóng)成蟲(chóng)總RNA為模板,經(jīng)反轉(zhuǎn)錄PCR(RT-PCR)擴(kuò)增目的編碼基因。純化PCR產(chǎn)物與pGEM-T載體連接后轉(zhuǎn)化感受態(tài)大腸桿菌JM109,抽提重組質(zhì)粒DNA用BamHⅠ和SalⅠ雙酶切及核苷酸序列測(cè)序進(jìn)行鑒定。選擇閱讀框正確的克隆,純化重組質(zhì)粒中目的基因雙酶切片段,亞克隆入pET28a原核表達(dá)載體,構(gòu)建重組質(zhì)粒pET28a-SjHMGB1,轉(zhuǎn)化DH5α感受態(tài)菌,重組質(zhì)粒經(jīng)雙酶切和核苷酸序列鑒定后,陽(yáng)性克隆質(zhì)粒轉(zhuǎn)化感受態(tài)大腸桿菌BL21(DE3),IPTG誘導(dǎo)表達(dá)并獲得純化的重組蛋白,采用SDS-PAGE和Western blotting分析和鑒定該重組蛋白。運(yùn)用Gene Runner軟件預(yù)測(cè)reSjHMGB1的二級(jí)結(jié)構(gòu)、功能位點(diǎn)及表位特征。在免疫保護(hù)性實(shí)驗(yàn)中,雌性C57BL/6小鼠隨機(jī)分為5組,分別為感染對(duì)照組、弗氏佐劑對(duì)照組、MontanideISA206佐劑對(duì)照組、reSjcHMGB1加弗氏佐劑免疫組、reSicHMGB1加MontanideISA 206佐劑免疫組。感染對(duì)照組不注射任何抗原和佐劑,兩種佐劑對(duì)照組小鼠注射乳化的生理鹽水加弗氏或Montanide ISA 206佐劑,兩免疫組每只小鼠經(jīng)背部皮下多點(diǎn)注射乳化的20μg reSjcHMGB1加弗氏或Montanide ISA 206佐劑,共免疫3次,間隔2周。末次免疫后2周,小鼠經(jīng)腹部感染日本血吸蟲(chóng)尾蚴30±1條,攻擊感染后6周剖殺小鼠,進(jìn)行成蟲(chóng)和蟲(chóng)卵計(jì)數(shù)。并分別于免疫前、攻擊感染前和小鼠剖殺前采血并分離血清,ELISA檢測(cè)血清中特異性IgG抗體。結(jié)果,RT-PCR擴(kuò)增的目的片段經(jīng)瓊脂糖凝膠電泳觀察,與預(yù)計(jì)的一致。TA克隆插入目的片段經(jīng)核苷酸序列測(cè)定,cDNA全長(zhǎng)531 bp,編碼176個(gè)氨基酸。序列分析表明該片段與SmHMGB1基因序列同源性為86%,推導(dǎo)的氨基酸序列同源性為93%。表達(dá)蛋白經(jīng)SDS-PAGE分析顯示,reSjHMG重組蛋白的分子質(zhì)量約30 kDa(包括6個(gè)組氨酸),以可溶性方式表達(dá)。免疫印跡結(jié)果顯示,日本血吸蟲(chóng)感染小鼠血清、重組抗原免疫小鼠血清和抗His-G HRP抗體均可識(shí)別該重組蛋白。生物信息學(xué)分析表明該蛋白包含兩個(gè)保守的結(jié)構(gòu)域(A盒和B盒)及含酸性氨基酸的C末端,同時(shí)存在多個(gè)潛在的抗原決定簇。在免疫保護(hù)性實(shí)驗(yàn)中,ELISA結(jié)果表明,免疫后重組抗原加兩種佐劑免疫組小鼠的特異性IgG抗體水平均顯著高于感染對(duì)照組和佐劑對(duì)照組(P＜0.05)。reSjcHMGB1加弗氏佐劑免疫組減蟲(chóng)率和肝組織減卵率分別為17.9%和17.6%,其蟲(chóng)荷數(shù)和每克肝組織蟲(chóng)卵數(shù)(EPG)與感染對(duì)照組相比均無(wú)統(tǒng)計(jì)學(xué)意義(P＞0.05)。reSjcHMGB1加Montanide ISA 206佐劑免疫組減蟲(chóng)率和肝組織減卵率分別為分別為33.2%和11.3%,其蟲(chóng)荷數(shù)與感染對(duì)照組相比有統(tǒng)計(jì)學(xué)意義(P＜0.05),但與ISA 206佐劑對(duì)照組相比無(wú)統(tǒng)計(jì)學(xué)意義(P＞0.05)。本研究成功克隆、表達(dá)SjHMGB1,并獲得純化的重組蛋白。在動(dòng)物保護(hù)性實(shí)驗(yàn)中,重組抗原并未誘導(dǎo)小鼠產(chǎn)生明顯的抗感染和抗生殖免疫保護(hù)作用。
[Abstract]:Schistosomiasis japonica, as a zoonotic parasitic disease, is still an important public health problem in China. At the present stage, the prevention and control strategy of schistosomiasis, which is based on the control of the source of infection and comprehensive treatment, is the basis for the prevention and treatment of schistosomiasis, but the treatment of the disease is the basis of the prevention and treatment of schistosomiasis. Schistosomiasis is still spreading to the new area, and the chemotherapy of praziquantel has certain limitations. Group chemotherapy does not prevent repeated infection and may produce the resistance strain of the praziquantel. Therefore, in the strategy of schistosomiasis control, the vaccine strategy has been considered to be an important supplement to the praziquantel treatment. The genes of a variety of vaccine candidate antigens have been cloned and expressed, and animal protection experiments have been carried out. Although a certain expected effect has been achieved, it is still an important direction to continue to search for new candidate antigens and improve the immunogenicity of the candidate vaccine molecules, and the cDNA text is screened by immunization. To find more effective vaccine candidates is an effective way to obtain new candidate antigen molecules.
The main purpose of the development of the vaccine is to reduce the deposition of worm and egg in the liver tissue, so the effect of the vaccine should be more aimed at the spawning of the adult and the adult. Microtus Fortis (Mf) is a mammalian animal that is not pathogenic after infection of Schistosoma japonicum. To find out the specific antigen molecules in the stage of the child's insect, eliminate the schistosomiasis at this stage or prevent its growth, development, maturation, spawning and pathogenicity, which can not only reduce the pathological damage caused by schistosomiasis, but also effectively prevent its transmission. Therefore, the cDNA Library of Schistosoma japonicum is screened by the immunization of the serum of voles orient in order to find the phase. These results may provide information for the study of the natural mechanism of resistance of Microtus Fortis to Schistosoma japonicum.
Arginine methylation plays an important role in the regulation of gene expression in Schistosoma. This is a post-translational modification that participates in a variety of cell functions, including RNA processing, cell signal transduction, protein subcellular localization, post transcriptional regulation and DNA repair.
High mobility group protein B1 (HMGB1) participates in gene transcription, replication, recombination and repair. Extracellular HMGB1 is an important late inflammatory mediator, which activates the release of early inflammatory factors such as TNF- A and IL-13 in macrophages. In Schistosoma infection, TNF- alpha and IL-13 play an important role in inducing the formation of buds around the eggs. .HMGB1, a key molecule of host immune regulation after infection, is closely related to the pathogenesis of some infectious diseases.
In this study, the positive clones screened from the cDNA Library of Schistosoma japonicum were selected to study the protein arginine methyltransferase 1 (PRMT1) and high mobility group protein B1 (HMGB1). First, the complete open reading frame was obtained by bioinformatics analysis, and then the 2 genes were introduced by molecular cloning technology. Subsequently, the purified recombinant protein reSjcHMGB1 was cloned and expressed in vivo, and its value as a vaccine candidate antigen was evaluated.
Immunological screening of genes related to natural resistance of Schistosoma japonicum in Microtus fortis
The cDNA Library of Schistosoma japonicum liver stage was screened using the natural resistance of Schistosoma japonicum resistance to the serum of voles Orient. The positive clones obtained from 3 times of rescreening were transformed into Escherichia coli (E.coil) BM25.8 cyclization plasmids, DNA, EcoR I and Hind III double enzyme cut agarose gel electrophoresis, and the inserted fragment was sequenced. Bioinformatics analysis. Results, 32 positive clones were obtained after 3 rescreening. The inserted fragments were 300 BP to 1100 BP. The sequencing results obtained 26 different molecular genes: high mobility group protein B1 (HMGB1) partial gene, protein arginine methyltransferase partial encoding gene, and cytochrome b encoding gene, Mitochondrial coding region gene, 16 unknown protein encoding gene of Schistosoma japonicum, and 6 unknown new genes of Schistosoma japonicum. This study uses the serum of vole vole to screen the cDNA Library of Schistosoma japonicum, and obtains a number of new coding genes for the candidate of Schistosoma japonicum vaccine, which lays a foundation for the study of schistosomiasis vaccine and schistosomiasis immunity diagnosis. Set the foundation.
Two, cloning, expression and analysis of the gene encoding protein arginine methyltransferase (PRMT) 1 of Schistosoma japonicum.
A pair of primers was designed based on the SjPRMT1 gene sequence obtained by the electron extension. The upstream primers introduced the BamH I enzyme cut site and the downstream primers introduced the Xho I enzyme cut site. The target encoding gene of the total RNA of Schistosoma japonicum was amplified by reverse transcriptional PCR (RT-PCR). The purified PCR product was connected with pGEM-T vector to convert the susceptible Escherichia coli JM109. The recombinant plasmid DNA was identified by BamH I and Xho I double enzyme digestion and nucleotide sequence sequencing. Select the correct clone of the reading frame, purify the double enzyme cut fragment of the target gene in the recombinant plasmid, subclone into the pET28a prokaryotic expression vector, construct the recombinant plasmid pET28a-SjPRMT1, transform the DH5 alpha receptive bacteria, and the recombinant plasmid through double enzyme cutting and nucleotides. After the sequence identification, the positive cloned plasmid transformed the receptive Escherichia coli BL21 (DE3), IPTG induced the expression and obtained the purified recombinant protein (referred to as reSjPRMT1). The recombinant protein was analyzed and identified by SDS-PAGE and Western blotting. The Gene Runner software was used to predict the two structure of the reSjPRMT1 protein, the functional site and the epitope characteristics. Results, RT. -PCR amplified a size and expected gene fragment.TA clones to be inserted into the target fragment by nucleotide sequence and cDNA was 1083 BP and encoded 360 amino acids. Sequence analysis showed that the fragment was homologous with the SmPRMT1 gene sequence of 87%. The deduced amino acid sequence homology was analyzed by SDS-PAGE and Western blotting analysis of 95%. expression protein. The results showed that the molecular mass of the recombinant protein of reSjPRMT1 was about 43 kDa (including 6 histidine), expressed in a soluble way, could be cloned by the sera of mice infected with Schistosoma japonicum and the anti His-G HRP antibody to identify the.SjPRMT1 gene. The expression was successful, and the purified recombinant protein was obtained. It could further study its biological characteristics and immunogenicity for the future. The foundation is laid.
Three, cloning, expression and immunological protection of high mobility group protein B1 (HMGB1) gene of Schistosoma japonicum.
A pair of degenerate primers was designed according to the published SmHMGB1 gene sequence. The upstream primers introduced the BamH I enzyme cutting site and the downstream primers introduced the Sal I enzyme cut site. The total RNA of the adult Schistosoma japonicum was used as the template, and the target encoding gene was amplified by the reverse transcription PCR (RT-PCR). The purified PCR product was connected with pGEM-T vector to convert the susceptible Escherichia coli JM109 and pumped. The recombinant plasmid DNA was identified by BamH I and Sal I double enzyme digestion and nucleotide sequence sequencing. Select the correct clone of the reading frame, purify the double enzyme cut fragment of the target gene in the recombinant plasmid, subclone into the pET28a prokaryotic expression vector, construct the recombinant plasmid pET28a-SjHMGB1, transform the DH5 alpha susceptible strain, and the recombinant plasmid through double enzyme cutting and nucleotide sequence. After identification, the positive cloned plasmid transformed the receptive Escherichia coli BL21 (DE3), IPTG induced expression and obtained the purified recombinant protein. The recombinant protein was analyzed and identified by SDS-PAGE and Western blotting. The Gene Runner software was used to predict the two stage structure of reSjHMGB1, the functional loci and the epitope characteristics. In the immunoprotective experiment, female C57BL/6. Mice were randomly divided into 5 groups: infection control group, Freund's adjuvant control group, MontanideISA206 adjuvant control group, reSjcHMGB1 Freund adjuvant immune group, reSicHMGB1 plus MontanideISA 206 adjuvant immune group. The infection control group was not injected with any antigen and adjuvant, two kinds of adjuvant injected phacoemulsification saline or Montan to mice. IDE ISA 206 adjuvant, two immunization groups were injected into the back subcutaneous injection of 20 mu g reSjcHMGB1 and Montanide ISA 206 adjuvant, immunized for 3 times, interval 2 weeks. After the last immunization, the mice infected with Schistosoma japonicum cercariae 30 + 1 weeks after the last immunization. The mice were killed 6 weeks after the infection, and the count of adults and eggs was carried out. And respectively. Before the immunization, the blood was collected before and before the infection of the infected mice and the serum was isolated, and the specific IgG antibody in the serum was detected by ELISA. Results the target fragment of RT-PCR amplification was observed by agarose gel electrophoresis, and the same.TA clone inserted into the target fragment was determined by nucleotide sequence. The total length of cDNA was 531 BP and encoded 176 amino acids. Sequence analysis showed that this The homology of the fragment and SmHMGB1 gene sequence was 86%. The deduced amino acid sequence homology was 93%. expression protein by SDS-PAGE analysis. The molecular weight of the recombinant protein of reSjHMG was about 30 kDa (including 6 histidine) and expressed in a soluble way. The result of immunoblotting showed that the mice sera infected by Schistosoma japonicum and the recombinant antigen were immune to the mice serum. And anti His-G HRP antibodies can identify the recombinant protein. Bioinformatics analysis shows that the protein contains two conservative domains (A box and B box) and the C terminal of acid amino acids, and there are several potential antigenic determinants. In the immunoprotective experiment, ELISA results show that the post immune recombinant antigen and two adjuvant immune groups are small. The level of specific IgG antibody in rats was significantly higher than that in the infection control group and the adjuvant control group (P < 0.05).ReSjcHMGB1 gf's adjuvant immune group and the liver tissue reduction rate were 17.9% and 17.6% respectively. The number of worms and the number of eggs per gram of liver tissue (EPG) were not statistically significant (P > 0.05).ReSjcHMGB1 plus Montanide (P > 0.05) and Montanide The rate of worm reduction and liver tissue reduction were 33.2% and 11.3% respectively in the ISA 206 adjuvant immunization group, respectively, and the number of parasite was statistically significant compared with the control group (P < 0.05), but it was not statistically significant compared with the ISA 206 adjuvant control group (P > 0.05). This study was successfully cloned, expressed SjHMGB1, and obtained the purified recombinant protein. In the experiment, recombinant antigen did not induce significant anti infection and anti reproductive immunity in mice.
【學(xué)位授予單位】：中國(guó)疾病預(yù)防控制中心
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類號(hào)】：R383.2

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