本文選題：日本血吸蟲 + sjHSP40�。� 參考：《南方醫(yī)科大學》2012年碩士論文

【摘要】：目的：構(gòu)建PGEX-6P-1-SjHSP40(SjHSP40)融合表達載體并在大腸埃希菌(Escherich coli內(nèi)進行原核表達及重組蛋白純化,初步探討SjHSP40的免疫學功能。 方法：首先,對SjHSP40進行生物信息學分析,以PCR方法擴增SjHSP40基因片段,將其克隆入原核表達質(zhì)粒pGEX-6P-1,經(jīng)測序驗證后轉(zhuǎn)化入E. coli BL-21(DE3)中。隨后,重組蛋白經(jīng)異丙基硫代半乳糖苷(IPTG)誘導表達后以Glutathione-Sepharose4B親和柱進行純化,所得產(chǎn)物GST-SjHSP40融合蛋白進行十二烷基磺酸鈉-聚丙烯酰胺凝膠電泳(SDS-PAGE)、Western blot鑒定。最后,將此融合蛋白免疫Balb/c小鼠后,以ELISA法檢測血清中SjHSP40特異性抗體水平(IgG及其亞型IgG1和IgG2a),并以流式細胞術(shù)通過體內(nèi)外實驗觀察SjHSP40對CD4+T淋巴細胞分化(包括Th1、Th2、Th17和Treg細胞)的動態(tài)變化。 結(jié)果：對SjHSP40進行生物信息學分析發(fā)現(xiàn),SjHSP40的氨基酸序列與曼氏血吸蟲的同系物相似性最高,序列一致性達到86%,與人、小鼠HSP40的氨基酸序列一致性分別為37%和39%,SjHSP40含有7個潛在的B細胞表位及多個T細胞表位。測序結(jié)果表明,本研究成功構(gòu)建了pGEX-6P-1-SjHSP40原核表達載體,經(jīng)IPTG誘導表達出融合蛋白GST-SjHSP40。Western blot結(jié)果顯示該融合蛋白能被抗GST抗體特異性識別。ELISA檢測表明,與GST等對照組相比,此融合蛋白免疫小鼠血清中抗原特異性IgG及其亞型IgGl和IgG2a水平均顯著升高。通過體內(nèi)外實驗,運用流式細胞術(shù)檢測顯示,與GST等對照組相比,本實驗中未見SjHSP40對CD4+T淋巴細胞分化的具有明顯影響。 結(jié)論：本實驗中SjHSP40可誘導免疫小鼠血清中高水平的特異性IgG,包括其亞型IgG1和IgG2a,但對CD4+T淋巴細胞的分化(包括Thl、Th2、Th17和Treg細胞)卻未見明顯影響。
[Abstract]:Aim: to construct a fusion expression vector PGEX-6P-1-SjHSP40 (SjHSP40) and express it in E. coli Escherich coli, and to investigate the immunological function of SjHSP40. Methods: firstly, SjHSP40 was analyzed by bioinformatics. The SjHSP40 gene fragment was amplified by PCR and cloned into prokaryotic expression plasmid pGEX-6P-1. The SjHSP40 gene was transformed into E. coli BL-21 (DE3) after sequencing. Then, the recombinant protein was induced by isopropylthione-Sepharose 4B affinity column and purified. The GST-SjHSP40 fusion protein was identified by SDS-PAGEG blot. Finally, after immunizing Balb / c mice with this fusion protein, The specific antibody level of SjHSP40 and its subtypes, IgG1 and IgG2a, were detected by Elisa. The dynamic changes of CD4 T lymphocyte differentiation (including Th1, Th2Th2Th17 and Treg cells) induced by SjHSP40 were observed by flow cytometry in vitro and in vivo. Results: bioinformatics analysis of SjHSP40 showed that the amino acid sequence of SjHSP40 had the highest similarity with the homologue of Schistosoma mansoni. The amino acid sequence consistency of mouse HSP40 was 37% and 39% respectively. SjHSP40 contained 7 potential B cell epitopes and multiple T cell epitopes. The results of sequencing showed that pGEX-6P-1-SjHSP40 prokaryotic expression vector was successfully constructed, and the fusion protein GST-SjHSP40 was induced by IPTG. The results of Western blot showed that the fusion protein could be specifically recognized by anti-GST antibody. Elisa showed that the fusion protein could be compared with GST and other control groups. The levels of antigen-specific IgG and its subtypes IgGl and IgG2a in serum of mice immunized with this fusion protein were significantly increased. In vitro and in vivo experiments, flow cytometry showed that SjHSP40 had no significant effect on CD4 T lymphocyte differentiation compared with GST and other control groups. Conclusion: SjHSP40 can induce high level of specific IgG in serum of immunized mice, including its subtypes IgG1 and IgG2a, but has no significant effect on the differentiation of CD4 T lymphocytes (including ThlnTh2Th17 and Treg cells).
【學位授予單位】：南方醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R383.24

【共引文獻】

相關(guān)期刊論文 前8條

1 馬廣鵬;張興中;;熱休克蛋白70在抗感染與免疫中的作用[J];湖北畜牧獸醫(yī);2011年12期

2 孫國華;吉成龍;楊建敏;王衛(wèi)軍;任利華;;刺參(Apostichopus japonicus)高溫脅迫應答基因應激表達特性的分析[J];海洋與湖沼;2013年01期

3 侯云峰;卜培莉;;多器官功能障礙綜合征患者血清熱休克蛋白70的變化及其臨床意義[J];山東大學學報(醫(yī)學版);2013年06期

4 王作鵬;林文軍;金曉蘭;陸麗君;朱珊麗;張麗芳;;沙眼衣原體熱休克蛋白60的B細胞表位預測及分析[J];溫州醫(yī)學院學報;2010年05期

5 李莎莎;徐湘婷;劉瑋;徐志鵬;張偉偉;李永;董瀟瀟;楊曉瑋;劉豐;王sブ，

本文編號：2035096