嵌合口蹄疫病毒中和表位的猪细小病毒衣壳蛋白免疫原性研究

发布时间：2018-06-01 04:31

本文选题：口蹄疫病毒 + 猪细小病毒　；参考：《甘肃农业大学》2017年硕士论文

【摘要】：口蹄疫对我国养殖业造成严重威胁,由于传统灭活疫苗存在病毒毒力返强、灭活不彻底等不安全因素,促使人们研究更加安全的口蹄疫疫苗。PPV-VLPs亚单位疫苗具有类似天然病毒的稳定性和免疫原性,不具有病毒核酸,没有感染性,因此具有很好的应用前景。本研究以P1代重组病毒为材料,研究重组病毒增值工艺,利用杆状病毒表达系统,研究重组蛋白最适表达条件及免疫原性,以期研发出廉价、高效、安全的PPV-VLPs亚单位疫苗。本文通过对比昆虫细胞悬浮培养和贴壁培养2种方式下的细胞形态,研究昆虫细胞的最佳培养方式;通过用血球计数板每隔24h进行细胞计数,绘制悬浮细胞复制动力学曲线,研究昆虫昆虫细胞生长周期。为了得到高滴度的P3代重组病毒,将P1代重组病毒转染昆虫细胞,PCR鉴定插入的外源基因是否丢失;Western-blotting检测重组蛋白表达及其反应原性;采用不同的MOI感染昆虫细胞,不同时间收获病毒,通过测定病毒度,研究重组病毒增值工艺。采用不同的MOI感染昆虫细胞,不同感染时间收获细胞沉淀,进行Western-blotting分析,用软件对比灰度值,研究重组蛋白最适表达条件。采用最佳重组蛋白表达条件感染大量的悬浮昆虫细胞,进行重组蛋白的大量表达,将粗纯的蛋白表达产物加佐剂乳化肌肉注射豚鼠,采集血清,用间接ELISA方法检测抗体水平。结果表明,(1)昆虫细胞的最佳培养方式是悬浮培养;(2)为了得到高滴度的P3代重组病毒,PCR鉴定插入的外源基因没有因为传代而丢失;(3)研究重组病毒增值工艺,得出最佳MOI是0.05个MOI,最佳感染时间是72h;(4)重组蛋白在昆虫细胞中获得较好的表达,Western-blotting分析结果表明重组蛋白的反应原性良好,同时进一步证明了插入的口蹄疫病毒中和表位的存在;(5)研究重组蛋白最适表达条件,Westernblotting分析结果表明最适条件是10个MOI,最佳感染时间72h;(6)间接ELISA方法检测结果表明,灭活疫苗组和空衣壳免疫组都产生了较低水平的FMDV中和抗体和较高水平的PPV特异性抗体。(7)用SPSS软件对间接ELISA结果进行统计学分析,进一步验证了PPV空衣壳免疫原性。
[Abstract]:Foot-and-mouth disease (FMD) poses a serious threat to the breeding industry in China. Because of the unsafe factors such as the virus virulence and inactivation of traditional inactivated vaccines, It is suggested that a more safe foot-and-mouth disease vaccine. PPV-VLPs subunit vaccine has the similar stability and immunogenicity of natural viruses, no viral nucleic acid and no infection, so it has a good prospect of application. In this study, P1 generation recombinant virus was used as the material, the recombinant virus value-added technology was studied, and the optimal expression conditions and immunogenicity of recombinant protein were studied by using baculovirus expression system, in order to develop a cheap, efficient and safe PPV-VLPs subunit vaccine. In this paper, by comparing the morphology of insect cell suspension culture with that of adherent culture, we studied the optimum culture method of insect cell, and plotted the dynamic curve of suspension cell replication by counting cells every 24 hours with blood cell counter. The growth cycle of insect cells was studied. In order to obtain the high titer P3 generation recombinant virus, the P1 generation recombinant virus was transfected into insect cells to identify whether the inserted foreign gene was missing or not, and to detect the expression of recombinant protein and its reactivity by Western-blotting. The virus was harvested at different time, and the technology of recombinant virus increment was studied by measuring the virus degree. The optimal expression conditions of recombinant protein were studied by using different MOI infected insect cells and harvested cells at different time of infection. Western-blotting analysis was carried out and the gray value of recombinant protein was compared with software. A large number of suspended insect cells were infected with the best recombinant protein expression condition. The crude protein expression product was injected into guinea pig muscle with adjuvant emulsification. The serum was collected and the antibody level was detected by indirect ELISA method. The results showed that the best culture method for insect cells was suspension culture. In order to obtain high titer P3 recombinant virus, the inserted foreign gene was not lost due to passage. The results of Western-blotting analysis showed that the optimal MOI was 0. 05 moi, and the best infection time was 72 hau 4). The results of Western-blotting analysis showed that the reactivity of the recombinant protein was good. Furthermore, the existence of neutralizing epitopes of the inserted foot-and-mouth disease virus (FMDV) was confirmed.) the optimal expression conditions of recombinant proteins were analyzed by Western blotting. The results of Western blotting showed that the optimal conditions were 10 moi and the best time of infection was 72hmHX6) indirect ELISA method was used to detect the recombinant protein. Both the inactivated vaccine group and the empty capsid immunization group produced lower levels of FMDV neutralizing antibody and a higher level of PPV specific antibody. The results of indirect ELISA were statistically analyzed by SPSS software, which further verified the immunogenicity of PPV empty capsid.
【学位授予单位】：甘肃农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.65

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