猪圆环病毒2型病毒样颗粒的制备及免疫原性评价

发布时间：2018-02-12 00:25

本文关键词： 猪圆环病毒2型毕赤酵母表达系统亚单位疫苗病毒样颗粒　出处：《西北农林科技大学》2017年硕士论文　论文类型：学位论文

【摘要】：断奶仔猪多系统衰弱综合征(Postweaning Multisystemic Wasting Syndrome,PMWS)主要是由猪圆环病毒2型(Porcine circovirus type2,PCV2)引起的以生长缓慢、高度呼吸困难、严重腹泻、黄疸、淋巴结极度肿大等为典型特征的疾病,多发猪群是6~16周龄的仔猪。我国自从2000年首次发现该病以来,现在已经有20多个省、自治区、直辖市发现猪群中有PCV2的感染,阳性率为21%~96%。同时,PCV2进入机体后,能够破坏机体免疫系统,导致免疫力降低,造成与其他疾病的混合感染。因此,每年由该病造成的经济损失非常巨大。目前该病没有有效的治疗方法,疫苗免疫已经被证明是最有效的防控途径。传统的灭活疫苗存在着病毒增殖滴度低、生产成本高等问题,同时该类疫苗也有灭活不彻底的风险,目前已经逐渐被亚单位疫苗取代,尤其是圆环病毒样颗粒(Virus-like particles,VLPs)疫苗。Cap蛋白由于是圆环病毒唯一的结构蛋白,自身能够组装成VLPs,并且免疫后可诱导针对PCV2的完全保护性免疫,因此成为圆环亚单位疫苗研究的首选抗原。本论文根据实验室分离鉴定的PCV Cap基因序列,按照酵母密码子偏好性,进行密码子优化,构建了Cap蛋白的酵母表达载体,实现其分泌表达及VLPs组装,并用小鼠模型进行动物试验评价免疫效果,结果如下:1.成功构建了重组质粒pPICZα-A-AHCap、pPICZα-A-AHCap-Fla、pPICZα-A-SUMO-AHCap和酵母表达菌株GS115-pPICZα-A-AHCap、GS115-pPICZα-A-AHCap-Fla、GS115-pPICZα-A-SUMO-AHCap。2.利用以上构建的酵母表达菌株,成功获得了重组蛋白AHCap、AHCap-Fla、SUMO-AHCap。3.透射电镜观察纯化后的重组蛋白,结果发现:融合了SUMO标签的Cap蛋白(SUMO-AHCap)没有VLPs的形成,N端缺失32个氨基酸的AHCap以及在C端融合flagellin佐剂的AHCap(AHCap-Fla)明显可以看到直径在20 nm左右的VLPs,并且重组蛋白AHCap形成的VLPs比AHCap-Fla多。4.将纯化的重组蛋白AHCap、AHCap-Fla定量后制备亚单位疫苗,免疫小鼠检测其免疫原性。间接ELISA结果显示试验组小鼠血液中均含有PCV2的特异性抗体和中和抗体,IFN-γ和IL-2表达量均升高,并且在攻毒后的小鼠血清中未检测到病毒,而免疫重组蛋白AHCap-Fla的抗体滴度和细胞因子(IFN-?、IL-2)分泌高于IFN-?、IL-2组,说明重组蛋白AHCap-Fla和AHCap具有较好的免疫原性,但是重组蛋白AHCap-Fla的免疫效果更好。综上,本文利用毕赤酵母表达系统表达的重组蛋白AHCap、AHCap-Fla都可自组装成VLPs,并都具有较好的免疫原性,以上结果为PCV亚单位疫苗的研发提供了新思路。
[Abstract]:Postweaning Multisystemic Wasting Syndrometic syndrome (PMWS) is mainly caused by porcine circovirus type 2 PCV2), which is characterized by slow growth, severe dyspnea, severe diarrhea, jaundice and extremely large lymph nodes. Since 2000, when the disease was first discovered in China, more than 20 provinces, autonomous regions and municipalities have found PCV2 infection in pigs, and the positive rate is 21%. At the same time, when PCV2 entered the body, It can destroy the body's immune system, reduce immunity, and cause mixed infections with other diseases. So the annual economic losses caused by the disease are enormous. There are no effective treatments for the disease. Vaccine immunization has been proved to be the most effective way of prevention and control. Traditional inactivated vaccines have problems such as low virus proliferation titer, high production cost and so on. At the same time, this kind of vaccine also has the risk of inactivation. Recently, it has been gradually replaced by subunit vaccine, especially the Virus-like particleso VLPs.Cap protein, which is the only structural protein of circovirus, can be assembled into VLPs by itself, and can induce complete protective immunity against PCV2 after immunization. In this paper, according to the sequence of PCV Cap gene isolated and identified in laboratory, according to the preference of yeast codon, we constructed the yeast expression vector of Cap protein. The recombinant plasmid pPICZ 伪 -A-AHCap-AHCap-pPICZ 伪 -A-AHCap-Fla-A-SUMO-AHCZ 伪 -A-SUMO-AHCap and the yeast expression strain GS115-pPICZ 伪 -A-AHCapGS115-pPICZ 伪 -A-APICZ 伪 -A-SUMO-AHCap.2were successfully constructed. The recombinant protein AHCapA AHCap-Flaa SUMO-AHCap.3.The purified recombinant protein was observed by transmission electron microscope. The results showed that the Cap protein (SUMO-AHCapp) with SUMO tag had no AHCap with 32 amino acids missing from the N-terminal of VLPs and the AHCap with flagellin adjuvant on the C-terminal. The AHCap with diameter of about 20 nm could be seen, and the recombinant protein AHCap could be formed. VLPs was more than AHCap-Fla. The purified recombinant protein AHCap-Fla was quantified and subunit vaccine was prepared. The immunogenicity of the immunized mice was detected by indirect ELISA. The results of indirect ELISA showed that the expression of PCV2 specific antibody and neutralizing antibody, IFN- 纬 and IL-2, were increased in the blood of the test group, and no virus was detected in the serum of the mice after the attack. The antibody titers and cytokines of the recombinant protein AHCap-Fla were determined. The secretion of IL 2 is higher than that of IFN? IL-2 group showed that the recombinant protein AHCap-Fla and AHCap had better immunogenicity, but the recombinant protein AHCap-Fla had better immunogenicity. In this paper, the recombinant protein AHCap-Fla expressed by Pichia pastoris can be self-assembled into VLPsand have good immunogenicity. These results provide a new idea for the development of PCV subunit vaccine.
【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】：2017
【分类号】：S858.28

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