风疹病毒（RV）包膜糖蛋白E1特异基因片段的原核表达

发布时间：2018-05-01 11:18

  本文选题：风疹病毒 + E1特异肽段　； 参考：《南京医科大学》2009年硕士论文

【摘要】：风疹病毒(rubellavirus,RV),又称德国麻疹病毒,是披膜病毒科风疹病毒属的唯一成员,与披膜科的其它病毒无交叉抗原。RV是TORCH综合征病原体(巨细胞病毒、单纯疱疹病毒、风疹病毒、弓形虫)之一,是重要的胎儿致畸因子,可以通过胎盘感染胎儿,导致新生儿患上先天性风疹综合征(congenitalrubellasyndrome,CRS),可表现为白内障、耳聋、心脏病或智力发育障碍。孕妇感染RV越早,新生儿患CRS的机率越大。RV也是男性不育的影响因子,男性生殖系统感染TORCH,可引起睾丸炎、附睾炎、前列腺炎等,以及男性不育。 目前,免疫学检测手段一直是广泛应用于RV感染的临床诊断方法,如血凝抑制试验(HIA),特异性抗体IgM、IgG及RV蛋白抗原的检测等。RV感染的筛查主要基于ELISA法检测特异性的血清IgM型抗RV抗体,检测试剂盒几乎完全依赖于进口,价格昂贵,很难适合于临床实验室常规开展。抗RV抗体检测结果的准确性依赖于相应的抗原制剂,因此,抗原的特异性决定了检测结果的准确性。尽管RV的主要三种结构蛋白——包膜糖蛋白E1、E2和衣壳蛋白C都有着较高的免疫原性,在人体内可诱发强烈的抗体应答。其中E1包括RV的大部分血凝抑制区和免疫抗原表位,且经研究表明E1基因序列株间变异程度与全株序列变异程度相近,而E2蛋白的生物学作用尚不清楚,衣壳蛋白C主要与病毒复制有关,后者因存在交叉抗体应答限制了其作为诊断的特异性抗原。因此,RV的3种结构蛋白中,E1蛋白最适合作为基因工程抗原用做免疫性诊断试剂和疫苗的研制。 本研究从麻疹、腮腺炎及风疹三联减毒活疫苗中提取了RV的基因组RNA,将其逆转录为cDNA,便于对其基因组保存及进一步扩增目的基因。而后通过基因工程方法表达E1基因保守性好、免疫原性高的氨基端196-305位氨基酸的肽段E1-N。用PCR扩增得到编码E1-N的基因片段,插入原核表达载体pGEX-2T质粒中,构建成表达E1-N的重组质粒pGEX-2T/E1-N,经双酶切和测序鉴定证实后,将该质粒转化E.coliBL21菌株,经异丙基-β-D硫代半乳糖苷(IPTG)诱导,分别于16℃和37℃下诱导表达融合蛋白,得到结果:在16℃诱导下表达的谷胱甘肽-S-转移酶(GST)与E1-N的融合蛋白主要存在于上清中;而37℃诱导下的融合蛋白主要存在于包涵体中。通过谷胱甘肽琼脂糖珠亲和层析获得纯化的GST/E1-N融合蛋白。 通过以上研究,我们扩增的包膜糖蛋白的E1特异基因序列,经测序证实与GenBank上所报道的基因序列一致,用IPTG诱导重组表达质粒pGEX-2T/E1-N表达重组融合肽段时,IPTG的最佳浓度为1mmol/L,最佳诱导时间为5h,最佳诱导温度为16℃。低温诱导可以避免包涵体的形成,重组融合蛋白以可溶性形式表达于上清中,从而避免了对重组融合蛋白的变性和复性过程,大大简化了操作。因此,本研究表达的E1特异蛋白为进一步研制特异性RV检测试剂盒和开发新型基因工程疫苗提供了实验依据和方法基础。
[Abstract]:Rubella virus, also known as rubella virus, is the only member of the rubella virus genus of the family Erythroviridae. RV is the pathogen of TORCH syndrome (cytomegalovirus, herpes simplex virus, rubella virus). Toxoplasma gondii), one of the important fetal teratogenic factors, can infect the fetus through the placenta and lead to congenital rubella syndrome (CCS), which can be characterized by cataract, deafness, heart disease or mental retardation. The earlier the pregnant woman infects RV, the greater the chance of neonatal CRS. RV is also the influence factor of male infertility. The male reproductive system infection with Torch can cause orchitis, epididymitis, prostatitis, and male infertility. At present, immunological detection has been widely used in the clinical diagnosis of RV infection. Such as hemagglutination inhibition test, detection of specific antibody IgG and RV protein antigen, and so on. The screening of RV infection is mainly based on ELISA method to detect the specific serum IgM type anti-RV antibody. The detection kit is almost completely dependent on imports, and the price is high. It is difficult to be used in clinical laboratory routine. The accuracy of anti-RV antibody detection depends on the corresponding antigens, so the specificity of antigens determines the accuracy of the detection results. Although the three main structural proteins of RV, the envelope glycoprotein E1E _ 2 and capsid protein C, have high immunogenicity, they can induce a strong antibody response in human body. Among them, E1 includes most of hemagglutination inhibition region and immune antigen epitope of RV. The results show that the variation degree of E1 gene sequence is similar to that of whole plant sequence, but the biological function of E2 protein is not clear. Capsid protein C is mainly associated with viral replication, which is restricted by cross-antibody responses as a specific antigen for diagnosis. Therefore, among the three structural proteins of RV, the E 1 protein is the most suitable for the development of immunological diagnostic reagent and vaccine as a genetic engineering antigen. In this study, the genomic RNAs of RV were extracted from live attenuated measles, mumps and rubella vaccine. Then we expressed the amino acid peptide E1-Nof the amino terminal of the amino terminal of 196-305 amino acid with high immunogenicity and good conservation of E1 gene by genetic engineering method. The gene fragment encoding E1-N was amplified by PCR and inserted into the prokaryotic expression vector pGEX-2T plasmid. The recombinant plasmid pGEX-2T / E1-N was constructed and confirmed by double enzyme digestion and sequencing. The plasmid was transformed into E.coliBL21 strain. The fusion protein was induced by isopropyl- 尾 -D-galactothioside (IPTG) at 16 鈩，

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