共表达PCV2 Cap蛋白和免疫增强因子InvC重组腺病毒甚因工程疫苗的研究

发布时间：2018-06-14 00:03

本文选题：猪圆环病毒2型 + Cap蛋白　；参考：《西北农林科技大学》2015年硕士论文

【摘要】：猪圆环病毒病(PCVDs)由猪圆环病毒2型(PCV2)引起,是当前对养猪业危害严重的传染性疾病。疫苗免疫接种是预防本病的主要手段,常用的疫苗有全病毒灭活疫苗,免疫效果较好,但存在病毒滴度较低、需要进行病毒浓缩、价格较高的问题。抗原滴度高、免疫效果好、使用更安全的PCV2疫苗研发是一个重要目的。腺病毒活载体疫苗具有制备相对简单、病毒滴度高、免疫刺激持久、效果确实等优点,在PCV2基因工程疫苗研究中受到关注。目前的重组病毒构建策略一般是将PCV2的衣壳蛋白(Cap)基因插入到病毒基因组中进行表达。假结核耶尔森菌侵袭素(Inv)是一种外膜蛋白,能够增强宿主细胞对抗原的摄取能力,提高疫苗免疫效果,蛋白的C端(InvC)在这一过程中发挥重要作用。本研究将PCV2的Cap基因和InvC基因在重组腺病毒共表达,构建一种同时表达目的抗原和免疫促进因子的重组腺病毒,进行动物免疫试验,对免疫效果进行评价,以期获得一种新型的PCV2腺病毒疫苗。本研究获得了以下结果:1.构建了共表达PCV2 Cap基因和InvC基因的重组腺病毒。将PCV2的Cap基因和InvC基因依次嵌入重组腺病毒穿梭载体,转化入BJ5183感受态细胞进行同源重组,获得重组腺病毒骨架质粒pAd-Cap-InvC,转染HEK293细胞,包装出重组腺病毒rAd-Cap-InvC。经PCR、RT-PCR、Western blot、IFA检测,rAd-Cap-InvC遗传基因稳定分析,证明Cap-InvC融合蛋白能够在重组病毒中得到表达。滴度可达109.32 TCID50/mL。2.重组病毒的动物免疫试验显示,其能有效刺激机体产生特异性的抗体。以108TCID50/只的重组腺病毒rAd-Cap-InvC和rAd-Cap分别免疫接种PCV2抗体阴性昆明系小鼠,首免后14 d加强免疫一次(108TCID50/只)。首免后14、21、35、42 d分别测定试验鼠血清中PCV2抗体水平。结果发现,两种重组腺病毒免疫小鼠均产生了针对PCV2 Cap的抗体,其中rAd-Cap-InvC免疫组比rAd-Cap免疫组的抗体滴度要高,在整个检测时间段均显著差异(P0.05)。以1010TCID50/只的重组腺病毒rAd-Cap-InvC,109TCID50/只的重组腺病毒rAd-Cap-InvC、rAd-Cap及rAd分别免疫接种试验猪2头,首免后14 d加强免疫一次(各组病毒剂量同首免)。首免后第14、21、28、36 d测定试验猪血清中PCV2抗体水平。结果发现,rAd-Cap-InvC组的抗体滴度在各检测时间点均要高于rAd-Cap组;1010TCID50/只的重组腺病毒rAd-Cap-InvC免疫组抗体滴度也高于109TCID50/只免疫组。动物免疫试验表明,重组腺病毒rAd-Cap-InvC和rAd-Cap均能使试验动物产生针对PCV2 Cap蛋白的特异性抗体,并且rAd-Cap-InvC能够产生更高的抗体滴度。本研究构建了共表达PCV2 Cap蛋白和免疫增强因子InvC重组腺病毒,其能使试验动物产生更高水平的特异性的免疫抗体,由此可知InvC对抗体的产生具有促进作用。
[Abstract]:Porcine circovirus disease (PCVDs) is caused by porcine circovirus type 2 (PCV2) and is a serious infectious disease which is harmful to pig industry at present. Vaccination is the main method to prevent this disease. The commonly used vaccine is the whole virus inactivated vaccine, the immune effect is good, but the virus titer is low, the virus concentration is needed, and the price is high. High antigen titer, good immune effect, the use of a more secure PCV 2 vaccine development is an important goal. Adenovirus live vector vaccine has many advantages, such as relatively simple preparation, high titer of virus, long-lasting immune stimulation and definite effect, so it has attracted much attention in the research of PCV2 gene engineering vaccine. Currently, the construction strategy of recombinant virus is to insert PCV2 capsid gene into the virus genome for expression. The involute of Yersinia pseudotuberculosis is an outer membrane protein, which can enhance the ability of the host cells to ingest antigen and improve the immune effect of the vaccine. The C terminal InvC of the protein plays an important role in this process. In this study, the Cap gene and InvC gene of PCV2 were co-expressed in recombinant adenovirus, and a recombinant adenovirus expressing both target antigen and immune promoting factor was constructed. In order to obtain a new type of PCV 2 adenovirus vaccine. The results of this study are as follows: 1: 1. A recombinant adenovirus co-expressing PCV2 Cap gene and InvC gene was constructed. The Cap gene and InvC gene of PCV2 were inserted into the recombinant adenovirus shuttle vector, and then transformed into BJ5183 cells for homologous recombination. The recombinant adenovirus skeleton plasmid pAd-Cap-InvCwas transfected into HEK293 cells, and the recombinant adenovirus rAd-Cap-InvCwas packaged. The stability analysis of rAd-Cap-InvC gene by PCR RT-PCR Western blotsimetry showed that Cap-InvC fusion protein could be expressed in recombinant virus. Titer can reach 109.32 TCID 50 / ml. 2. Animal immunoassay of recombinant virus shows that it can effectively stimulate the body to produce specific antibodies. Recombinant adenovirus rAd-Cap-InvC and rAd-Cap were inoculated into mice with negative PCV2 antibody respectively. The serum levels of PCV2 antibody in mice were measured on day 14, 21 and 3 542 after the first immunization. The results showed that both recombinant adenovirus immunized mice produced antibodies against PCV2 Cap. The titer of antibody in rAd-Cap-InvC immunized group was higher than that in rAd-Cap immunization group, and the difference was significant in the whole detection period. The recombinant adenovirus rAd-Cap-InvCID50 / rAd-Cap-InvCnrAd-Cap and rAd were inoculated with two pigs respectively. Serum PCV2 antibody levels were measured at day 14, 21, 28 and 36 after the first immunization. The results showed that the titer of antibody in rAd-Cap-InvC group was higher than that in rAd-Cap group (1010TCID50 / rAd-Cap group) and 109TCID50 / mouse group. Animal immunoassay showed that both the recombinant adenovirus rAd-Cap-InvC and rAd-Cap could produce specific antibodies against PCV2 Cap protein, and rAd-Cap-InvC could produce higher antibody titers. In this study, co-expression of PCV2 Cap protein and immune enhancer InvC recombinant adenovirus was constructed. The recombinant adenovirus can produce a higher level of specific immune antibody in experimental animals, so InvC can promote the production of antibodies.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S855.3

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