丙型肝炎病毒高变区1模拟表位的交叉反应性与反应频率分析

发布时间：2018-06-21 12:51

本文选题：丙型肝炎病毒 + 高变区1　；参考：《第二军医大学》2007年硕士论文

【摘要】： 丙型肝炎病毒(hepatitis C virus,HCV)为黄病毒科丙型肝炎病毒属内唯一成员,其基因组是单股正链RNA。HCV感染可导致慢性丙型肝炎、肝硬化和肝细胞癌等,对人类健康造成严重危害。目前全球约有1.7亿HCV感染者,其中我国约有4000万。联合应用干扰素和利巴韦林是目前治疗HCV感染的标准方案,但治疗费用高,副作用较大,且疗效因HCV基因型别的不同而呈现明显差异。当前HCV疫苗研制尚未取得成功,筛选有效的抗原表位对于HCV疫苗的研发至关重要。位于病毒体表面的HCV包膜蛋白2(E2)区含有中和抗体表位,能诱导产生保护性抗体。高变区1(hypervariable region 1, HVR1)位于E2的N端,由27个氨基酸残基组成,序列高度变异, HVR1诱生的抗体无论在体外还是在黑猩猩的实验中均可以阻断HCV的感染。曾认为HVR1的变异使得抗体缺乏普遍保护性,然而最近的研究结果显示,一些不同氨基酸序列的HVR1具有明显的交叉抗原性,诱生的抗体具有交叉反应性。这为基于HVR1或E2蛋白的HCV疫苗的研制带来了希望。研究表明,用不同于天然HVR1氨基酸序列的合成肽免疫小鼠所诱导的抗体能与不同株HCV的HVR1发生交叉反应,这类能模拟来源于不同株HVR1抗原性的表位(模拟表位)在HCV疫苗研究中的应用前景值得探讨。一、HVR1融合蛋白在大肠杆菌中的表达、纯化及与HCV阳性血清的反应本研究分别从文献资料中选取了HVR1的12条模拟表位,将其编码序列与pET32a原核表达载体中的硫氧还蛋白(Trx)基因融合后插入pET32a载体中,构建的12种表达载体经琼脂糖凝胶电泳、酶切鉴定以及DNA测序证明扩增、拼接序列与预期完全一致。将12种重组表达质粒分别转化大肠杆菌BL21(DE3),用IPTG诱导目的蛋白表达,菌体经超声破碎后进行镍柱纯化,得到了12种HVR1融合蛋白。此外,还表达、纯化了空载体pET32a表达的硫氧还蛋白。收集30份HCV抗体阳性血清,10份正常对照血清,ELISA检测HVR1融合蛋白与血清的反应。各表位蛋白与10份健康人对照血清反应的OD值均低于0.1。各表位蛋白与30份阳性血清中的大部分能发生反应,反应率分别为46.7%(P1)、43.3%(P2)、66.7%(P3)、56.7%(P4)、70%(P5)、70%(P6)、53.3%(P7)、56.7%(P8)、70%(P9)、60%(P10)、60%(P11)、53.3%(P12)。这12种表位蛋白联合,可与30份血清中的29份发生阳性反应,反应率升至96.6%。进一步分析发现,P5、P6、P7、P9四种表位蛋白也联合可达到上述效果。其中P5、P6、P9均能与30份血清中的21份发生阳性反应,较其他9种表位蛋白为高。二、HVR1融合蛋白免疫小鼠及小鼠血清中的抗体检测。取上述HVR1融合蛋白以及空载体pET32a表达的硫氧还蛋白分别免疫BALB/c小鼠。融合蛋白与福氏完全佐剂乳化,背部皮下多点注射,200μg/只;2周后,肽与不完全福氏佐剂乳化,以相同剂量免疫,1次/2周,共2次。第8周取小鼠血清进行Western blotting和ELISA检测。 Western blotting检测发现,HVR1融合蛋白免疫的BALB/c鼠血清均可与相应的表位蛋白发生反应,表明血清中存在相应的抗表位蛋白的抗体。由于ELISA检测证实P5与丙肝阳性血清的强阳性反应率最高,所以我们合成了P5肽,ELISA方法分析P5肽与12种HVR1融合蛋白免疫BALB/c鼠血清的反应。结果:合成P5肽与其他11种HVR1融合蛋白免疫BALB/c鼠血清可发生不同程度的交叉反应。与硫氧还蛋白免疫的鼠血清不发生反应。 ELISA检测HVR1融合蛋白免疫的BALB/c鼠血清与HVR1合成肽的反应频率。12种表位蛋白免疫的鼠血清均能与2条具有较大差异J4和H77株的HCV HVR1表位合成肽发生反应。空载体pET32a表达的硫氧还蛋白免疫BALB/c鼠血清并不与2条HVR1合成肽发生反应。此结果提示,12种表位蛋白均可诱导出与HCV HVR1表位合成肽具有高交叉频率的中和抗体。三、HCV假病毒中和试验将H77株pcDNA-HCVE1E2、pCMV-gag-pol、pCMV-GFP转染HEK293T细胞包装HCVpp,将含HCVpp的上清感染人肝癌Huh7.5细胞,感染52 h后,于荧光显微镜下观察GFP荧光。结果显示,三种质粒共转染HEK293T细胞上清感染的Huh7.5细胞有GFP绿色荧光的表达,表明HEK293T细胞上清中存在感染性的HCV假病毒;含假病毒的上清与P5、P6融合蛋白免疫鼠血清共孵育后感染7.5细胞,则GFP荧光明显减弱,荧光细胞数量减少。而含假病毒的上清与Trx免疫鼠血清共孵育后感染Huh7.5细胞,GFP荧光不受影响,表明HVR1融合蛋白免疫的鼠血清能中和HCVpp的感染性。结论:实验对12条HCV HVR1模拟表位进行研究,得到的表位蛋白能被多数HCV患者阳性血清特异识别,接种小鼠后能诱导出针对不同株HVR1的交叉反应性抗体,能中和HCVpp的感染性,提示用HVR1模拟表位可诱导具有交叉反应性的HCV中和抗体,因而在HCV疫苗研制中可能具有潜在的应用价值。
[Abstract]:Hepatitis C virus ( HCV ) is the only member of hepatitis C virus in the family Flaviviridae , and its genome is single strand of positive strand RNA . HCV infection can lead to chronic hepatitis C , liver cirrhosis and hepatocellular carcinoma . There are about 40 million HCV infection in China . There are approximately 170 million HCV infected persons in the world . The combination of interferon and ribavirin is the current standard for the treatment of HCV infection , but the therapeutic cost is high , the side effect is large , and the curative effect is different from the HCV genotype . The development of the current HCV vaccine has not been successful , and the screening of effective epitope is essential for the development of HCV vaccine .

The results showed that HVR1 induced by HVR1 could cross - react with HVR1 of different strains of HCV . The results showed that HVR1 induced by HVR1 could cross - react with HVR1 of different strains of HCV .

One , the expression of HVR1 fusion protein in E . coli , purification and reaction with HCV positive serum were used to select 12 mimic epitope of HVR1 from the literature and then inserted into pET32a vector . The 12 expression vectors were transformed into pET32a vector by agarose gel electrophoresis , enzyme digestion and DNA sequencing . Twelve recombinant expression plasmids were transformed into E . coli BL21 ( DE3 ) .

There were 30 sera of HCV antibody positive serum and 10 normal control serum , and the reaction of HVR1 fusion protein with serum was detected by ELISA . The reaction rates were 46.7 % ( P1 ) , 44.3 % ( P2 ) , 66.7 % ( P6 ) , 55.3 % ( P7 ) , 56.7 % ( P8 ) , 70 % ( P6 ) , 55.3 % ( P7 ) , 65.7 % ( P8 ) , 75.3 % ( P12 ) .

2 . Antibody detection of HVR1 fusion protein in mice and mouse serum .

BALB / c mice were immunized with the above HVR1 fusion protein and the expression of the empty vector pET32a , respectively . The fusion protein was emulsified with Fu ' s complete adjuvant and injected at 200 渭g / 2 for 2 weeks . After 2 weeks , the peptide was immunized with the adjuvant without complete Fu ' s adjuvant at the same dose for 2 times . Western blotting and ELISA were performed on the serum of mice at week 8 .

Western blotting showed that the serum of BALB / c mice immunized with HVR1 fusion protein could react with the corresponding epitope protein , indicating that there was corresponding anti - epitope protein in serum .

The results showed that the serum of BALB / c mice immunized with P - 5 - peptide and eleven HVR1 fusion proteins could cross - react in different degrees .

The serum of BALB / c mice immunized with HVR1 fusion protein and HVR1 synthesized peptide were detected by ELISA . All the 12 epitopes were able to react with two HCV HVR1 epitopes with a larger difference of J4 and H77 . The expression of the empty vector pET32a showed that the serum of BALB / c mice did not react with 2 HVR1 synthetic peptides .

III . neutralization test of HCV pseudovirus

H77 strains pcDNA - HCVE1E2 , pCMV - gag - pol , pCMV - GFP were transfected into HCVPs , and the supernatant was infected with human hepatoma Huh7.5 cells . After 52 h , GFP fluorescence was observed under fluorescence microscope .

The results showed that the expression of GFP green fluorescence in Huh7.5 cells co - transfected with three plasmids showed that infectious HCV pseudoviruses were found in the supernatant , and 7.5 cells were infected with the supernatant of the pseudovirus and the immunized mice with P5 and P6 .

Conclusion : 12 HCV HVR1 epitopes were studied . The results showed that the epitope protein can be specifically identified by the positive serum of most HCV patients . After inoculation of the mice , the cross - reactive antibodies against HVR1 of different strains can be induced , and the HCV neutralizing antibody with cross - reactivity can be induced by HVR1 analog epitope . Therefore , it is possible to have potential application value in the development of HCV vaccine .
【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R373

【参考文献】