丙型肝炎病毒多表位抗原基因pcx3的构建、表达及免疫原性研究

发布时间：2018-06-12 04:33

本文选题：多表位抗原 + 丙型肝炎病毒　；参考：《复旦大学》2005年博士论文

【摘要】：丙型肝炎病毒(HCV)为单股正链RNA病毒，其基因组编码约3010～3033aa的蛋白前体，从N-端起依次为核心蛋白(C)、包膜蛋白(E)、非结构蛋白NS1-NS5B。HCV具有高度的变异性，能够迅速突变逃逸宿主的免疫系统。因此，给疫苗的研究带来许多的困难。近年来的研究提示理想的HCV疫苗必须能够诱发高水平、广谱而特异的体液免疫应答和细胞免疫应答。针对HCV的高度变异性及免疫特点，在HCV基因产物中优选了5个具有良好免疫原性的高度保守B／T细胞表位，组合成一个多表位抗原基因pcx，这些表位分别来自核心蛋白(1～16aa，132～141aa)，E1(126～135aa)，NS3(139～147aa)及NS5B(768～775aa)，以及破伤风毒素的T细胞表位用以增强T细胞免疫。前期的研究表明与β-半乳糖苷酶融合表达的多表位抗原PCX具有良好的HCV免疫特异性。 pcx基因经过串联重复之后，构建成多表位抗原基因pcx3，与6×His-tag融合后，在E.coli宿主中获得高效表达。利用C端融合的6×His-tag经过Ni~(2+)-NTA-agarose亲和层析柱纯化后，得到高纯度的目的蛋白。利用BCA法对蛋白质进行定量。 Western blot结果显示原核或真核表达的PCX3抗原均能被HCV患者阳性血清所识别，表明多表位抗原具有能被HCV抗体特异性识别的表位。ELISA结果显示来自不同地区的不同患者血清中抗-HCV抗体能与PCX3抗原特异反应，62份阳性血清中PCX3抗原检测的检出率为83.8％，提示PCX3抗原的B细胞表位能被HCV抗体广泛识别。而PCX抗原的检出率为70％，表明经过串联重复后的抗原与HCV抗体的反应更强，显示出良好的免疫原性。纯化的PCX3蛋白与完全弗氏佐剂一起免疫BALB／c小鼠后，能够诱发高滴度的特异性IgG(1×10~6)，并持续保持数月。不同剂量组进行比较，发现50μg的剂量能够诱发理想的体液免疫反应。另外，利用PCX免疫小鼠的特异性抗体滴度最高达1×10~4，这一结果表明，与PCX抗原相比，串联重复的PCX3抗原能够诱发更强的免疫反应。抗PCX3抗血清与多肽的反应显示位于核心蛋白的P1(MTSTNPKPQRKTKRNTN)以及NS3蛋白的P6(TGDFDSVID)这两个B细胞表位与抗体反应的滴度较高，提示二者在诱导HCV特异的体液免疫反应中发挥主要作用。 PCX3蛋白可诱发较高水平的特异性CTL效应，特异性杀伤率为39.2％。在多肽表位中，NS5B的T细胞表位(ELITSCSS)的溶解率最高，提示该表位在诱发CTL杀伤效应中具有重要的作用。流式细胞仪检测外周血淋巴细胞表明，HCV多表位抗原PCX3免疫小鼠6周后，CD3~+CD4~+T细胞比例明显升高，说明该蛋白可以有效刺激CD3~+CD4~+Th细胞增殖。针对免疫小鼠的安全性评价表明这一多表位抗原免疫是安全而有效的。利用实时荧光定量PCR技术检测PCX3抗血清体外与病毒粒子(genotype 2a)的结合，实验结果表明孵育过夜后，PCX3抗血清在体外能够结合绝大部分的病毒粒子，初步显示PCX3抗血清具有抗病毒感染的可能，为进一步抑制病毒感染的试验提供基础。本实验中用于检验体液免疫被动保护反应的模型是SCID／Alb-uPA转基因小鼠。这种SCID／Alb-uPA纯合的小鼠体内进行人体肝细胞移植后，建立具有嵌合的人肝组织的转基因小鼠。利用HCV阳性血清进行腹膜内注射感染，病毒不仅能够复制，而且产生了具有感染性的颗粒，可以作为HCV感染的小鼠模型。利用该模型的研究表明HCV阳性血清(HCV genotype la，3.0E+6 IU／ml)与PCX3诱导的高滴度抗血清孵育过夜后进行感染，两周后检测发现实验组中有两只小鼠血清中含有低于500U的RNA拷贝数，其它三只血清中的RNA则为阴性，而对照组的7只小鼠全部感染了HCV，血清中的RNA拷贝数为3.0E+03到3.5E+06 IU／ml。这一结果表明PCX3诱导的抗血清能使2／5的小鼠病毒滴度显著减少，并且保护3／5的小鼠避免HCV感染，在被动免疫保护反应方面发挥重要的作用。对PCX3抗血清进行分析，发现它能与HCV的核心蛋白、NS3蛋白特异性结合，验证了抗血清中含有抗核心蛋白与NS3蛋白的抗体。利用PCX3抗原制备的单克隆抗体中筛选到两株分别与HCV核心蛋白以及NS3蛋白结合，一株与包膜蛋白E1的表位多肽P5(RMAWDMMMW)反应的单抗。表明PCX3诱导的体液免疫反应中包含这三个蛋白的B细胞表位(P1，P5和P6)所引起的免疫反应，且这些单抗可用于进一步研究抗体与病毒粒子的结合。在此基础上，选取抗核心蛋白的杂交瘤细胞株进行抗体的基因克隆，以期获得具有应用价值的单链抗体，结果筛选到表达特定条带的阳性克隆株，并经过测序鉴定。为抗体的应用研究奠定基础。综上所述，我们的结果表明，本研究所构建的多表位抗原PCX3可以诱导小鼠产生特异性细胞和体液免疫应答，，产生的抗体不仅能在体外结合基因型2a的HCV病毒粒子，且更重要的是，在小鼠的感染模型中抑制了基因型1a的HCV感染，因此，这一抗原有望成为广泛而有效的HCV疫苗候选者。
[Abstract]:Hepatitis C virus (HCV) is a single strand positive chain RNA virus, whose genome encodes about 3010 to 3033aa protein precursors, from the N- terminal to the core protein (C), the envelope protein (E), and the non structural protein NS1-NS5B.HCV with high variability, which can quickly mutate the immune system of the host and bring many difficulties to the vaccine research. Studies in recent years suggest that the ideal HCV vaccine must be able to induce high level, broad spectrum and specific humoral immune responses and cellular immune responses.
In view of the high variability and immunity of HCV, 5 highly conserved B / T cell epitopes with good immunogenicity were selected to form a multi epitope antigen gene PCX, which were derived from core proteins (1 to 16AA, 132 to 141aa), E1 (126 to 135AA), NS3 (139 147aa) and NS5B (768 to 768), and broken wounds, respectively. The T cell epitopes of the wind toxin are used to enhance the immunization of T cells. Previous studies have shown that the multi epitope antigen PCX expressed with beta galactosidase has a good HCV specific immune specificity.
After the PCX gene was repeated in series, the gene pcx3 was constructed into a multi epitope antigen gene. After the fusion with 6 x His-tag, the gene was highly expressed in the E.coli host. The purified target protein was obtained by the purification of the Ni~ (2+) -NTA-agarose affinity chromatography column with the fusion of C terminal 6 * His-tag. The protein was quantified by BCA method.
The Western blot results showed that the PCX3 antigen expressed in the prokaryotic or eukaryotic cells could be identified by the positive serum of the HCV patients, indicating that the multi epitope antigen has the epitopes that can be identified by the HCV antibody specific. The anti -HCV antibody in different patients from different regions can respond to the specific reaction of the -HCV antibody to the PCX3 antigen, and the PCX3 antigen in the 62 positive sera is detected. The detection rate was 83.8%, suggesting that the B cell epitope of PCX3 antigen was widely recognized by HCV antibody, while the detection rate of PCX antigen was 70%, indicating that the antigen after tandem repeats was more responsive to the HCV antibody and showed a good immunogenicity.
The purified PCX3 protein was immune to the BALB / c mice with the complete Freund adjuvant and could induce a high titer specific IgG (1 x 10~6) for a few months. Compared with the different dose groups, it was found that the dosage of 50 mu g could induce the ideal humoral immune response. In addition, the specific antibody of mice was immunized with PCX.
The highest titer was up to 1 x 10~4, which showed that the tandem repeat PCX3 antigen could induce a stronger immune response compared to the PCX antigen. The reaction of anti PCX3 antiserum to the polypeptide showed that the P1 (MTSTNPKPQRKTKRNTN) in the core protein and the P6 (TGDFDSVID) of the NS3 protein (TGDFDSVID), the two B cell epitopes of the NS3 protein, were higher in the titer of the antibody reaction, suggesting two It plays a major role in inducing HCV specific humoral immune response.
PCX3 protein can induce a high level of specific CTL effect, the specific killing rate is 39.2%. in the peptide epitopes, and the T cell epitopes of NS5B (ELITSCSS) have the highest dissolution rate, suggesting that the epitope plays an important role in inducing the killing effect of CTL. Flow cytometry shows that the HCV polyepitope antigen is immune to 6 mice in the peripheral blood gonorrhea cells. After week, the proportion of CD3~+CD4~+T cells increased significantly, indicating that the protein could effectively stimulate the proliferation of CD3~+CD4~+Th cells. The safety evaluation of immunized mice showed that the immunization of this multi epitope antigen was safe and effective.
Real-time fluorescence quantitative PCR was used to detect the combination of PCX3 antiserum with virus particles (genotype 2a) in vitro. The experimental results showed that after incubation for the night, the PCX3 antiserum could combine with most of the virus particles in vitro, and preliminarily showed that the antisera of PCX3 had the ability of antiviral infection, which provided a basis for the further inhibition test of virus infection.
In this experiment, the model used to test the passive protective reaction of humoral immunity is SCID / Alb-uPA transgenic mice. After transplantation of human hepatocytes in the SCID / Alb-uPA homozygous mice, transgenic mice with chimeric liver tissue were established. The virus can be injected intraperitoneally with HCV positive serum, and the virus can not only be replicated, but the virus can not only be copied, but also the virus can be replicated. The infectious particles could be produced as a mouse model of HCV infection. The study of the model showed that the HCV positive serum (HCV genotype La, 3.0E+6 IU / ml) was incubated with the high titer antiserum induced by PCX3 after night, and two weeks later, two mice in the experimental group were found to contain a RNA copy of less than 500U. The number of RNA in the other three sera was negative, while all the 7 mice in the control group were infected with HCV, and the RNA copy number in the serum was 3.0E+03 to 3.5E+06 IU / ml.. The results showed that the PCX3 induced antiserum could significantly reduce the titer of 2 / 5 mice, and protected 3 / 5 mice from the HCV infection, in the passive immune protection response side. The surface plays an important role.
The antiserum of PCX3 was analyzed. It was found that it could combine with the core protein of HCV, NS3 protein specifically, and verified the antibody containing anti core protein and NS3 protein in the antiserum. Two strains of monoclonal antibodies prepared by PCX3 antigen were selected to bind to HCV core protein and NS3 protein respectively.
The monoclonal antibody to the epitope peptide P5 (RMAWDMMMW) reaction of the envelope protein E1. It shows that the PCX3 induced humoral immune response includes the immune responses caused by the B cell epitopes of these three proteins (P1, P5 and P6), and these monoclonal antibodies can be used to further study the combination of antibodies and viral particles. On this basis, the hybridoma cells with anti core proteins are selected. To clone the antibody gene in order to obtain the applied value of single chain antibody, the positive clones expressing the specific bands were screened and identified by sequencing, which laid the foundation for the application of antibodies.
To sum up, our results show that the multi epitope antigen PCX3 constructed in this study can induce specific cell and humoral immune responses in mice. The produced antibodies not only combine the HCV virus particles of the genotype 2a in vitro, but also, more importantly, inhibit the HCV infection of the genotype 1a in the mouse infection model, thus, this Antigen is expected to become a widely effective candidate for HCV vaccine.
【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2005
【分类号】：R392

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