【作者】 王瑞；

【导师】 周斌； 李彬；

【作者基本信息】 南京农业大学 ， 兽医硕士（专业学位）， 2023， 硕士

【摘要】 猪轮状病毒（Porcine rotavirus,PoRV）在病原学分类中属于呼肠孤病毒科,轮状病毒属,是一种双链RNA病毒。轮状病毒感染动物可以使动物出现呕吐、腹泻、脱水、厌食等症状,由于轮状病毒感染通常伴随着其他腹泻病毒的共同感染或细菌的混合感染,在全世界范围内都造成了较大的经济损失,近年来,随着我国生猪养殖业的不断扩大,轮状病毒对生猪养殖业造成的损失也在扩大,在我国东北、西北、华北、华中、华南和西南等地区,轮状病毒发病率达到了40%以上,说明轮状病毒在我国呈现广泛流行的状态。由于轮状病毒目前并没有特效药,成年猪群的治疗和防控较为轻松,但针对仔猪的治疗和防控则更为重要,只有对仔猪进行安全有效的疫苗注射,才能降低仔猪的发病率和死亡率,在源头减少轮状病毒造成的损失。病毒样颗粒在蛋白的形态上与活病毒本身类似,但是不包含活病毒中的核酸物质,同时由于病毒样颗粒是由多种结构蛋白共同形成的,因此理论上这种多亚单位蛋白对动物机体的免疫原性要高于单一的亚单位蛋白,且在空间形态上与病毒类似,因此具有良好的免疫原性和反应原性,国外已经有成功组装VLPs的研究报道,且在疫苗研究方面都取得了不错的进展,为本实验提供了可行的思路。本实验针对轮状病毒的两种疫苗类型进行研究和探索,在传统疫苗方面尝试通过传代致弱的方法,为研发新型的轮状病毒减毒活疫苗奠定了基础。在新型疫苗方面,尝试在真核细胞平台上,对轮状病毒的四种重要的结构蛋白进行组装,最终构建出由不同结构蛋白共同形成的病毒样颗粒,为后续的轮状病毒病毒样颗粒疫苗的研发奠定了基础。具体研究如下:1.NJ2012传代突变分析轮状病毒会引起仔猪的严重腹泻,虽然亚单位疫苗或病毒样颗粒疫苗具有更好的免疫效果,但是其免疫注射的副作用相较于减毒活疫苗也相对较大,部分情况下可能并不适用,而我国的轮状病毒减毒活疫苗相关研究又较为缺少,为了研究轮状病毒减毒活疫苗,对NJ2012毒株进行连续传代至P56代,传代过程中每隔10代检测一次病毒滴度,NJ2012 P10/P20/P30/P40的病毒滴度分别达到10^7.6TCID50/mL、10^7.8TCID50/mL、10^8.2TCID50/mL以及10^8.3TCID50/mL,结果显示病毒的滴度在随着传代次数的增加而依次缓慢增加。将P30和P40的病毒液提取RNA后进行PCR扩增,成功扩增出大小约为2700bp、2000bp、1500bp、1000bp左右的VP2/VP4/VP6/VP7目的条带,将扩增后的产物经T载体连接处理后进行测序,测序结果显示P30和P40代次在VP2、VP4、VP6和VP7四个基因上出现了点突变、插入突变和缺失突变,由点突变引发了部分的氨基酸突变,没有出现大片段的核苷酸缺失或氨基酸缺失。通过使用DNASTAR和NCBI,选取GenBank上具有代表性的几种毒株的VP2、VP4、VP6、VP7全基因序列,与P30和P40相对应的基因序列进行对比,发现传代后的NJ2012-P30/P40与其他毒株的同源性可以达到70-99.9%之间,其中VP2基因与OSU毒株同源性最高,为99.5%,VP4基因与OSU-C5111毒株同源性最高,P30和P40与其同源性分别为83%和95.4%,VP6基因与K71,OSU等毒株同源性都很高,P30和P40与其同源性分别为95.2%和95.5%,VP7基因与HBYC/2015/G9等毒株同源性高达99.9%。P30和P40代次的毒株氨基酸同源性要低于核苷酸同源性,说明其突变过程中带来的氨基酸改变较多。2.猪轮状病毒VLPs的形成为了探索轮状病毒毒株NJ2012的四种结构蛋白VP2/4/6/7是否能够组装成功形成病毒样颗粒,如何组装,以及组装后的结果如何,参考了NCBI上已经发表的轮状病毒毒株NJ2012 VP2/4/6/7全长基因,分别针对这四个基因进行引物设计,合成了具有酶切位点和His标签的四对引物,使用这四对引物,以NJ2012为模板,成功扩增出VP2/4/6/7的全长基因。将已经扩增出的基因分别克隆到PMD-19T载体中,筛选阳性重组质粒后进行测序,测序结果显示扩增后的基因与NJ2012 VP2/4/6/7的核苷酸同源性分别达到了99.7%、98.6%、99.8%和99.9%,且核苷酸突变均为点突变,所产生的点突变对翻译后的氨基酸并没有产生影响,同时所需要的酶切位点及His标签均扩增成功。以鉴定后的阳性质粒为模板,扩增目的片段后,将四种目的基因分别克隆到真核细胞表达载体PCAGGS的EcoRⅠ、XhoⅠ和KpnⅠ、XhoⅠ之间的多克隆位点,经过PCR、单酶切和双酶切鉴定后,将成功构建的重组质粒转化至trans5α感受态细胞中,扩大培养四种重组质粒,分别命名为PCAGGS-VP2/4/6/7。将质粒单独转染293T细胞后,使用Western-blot和间接免疫荧光的方法,成功鉴定了四种质粒在293T细胞上的表达。分别将PCAGGS-VP2/4/6、PCAGGS-VP2/6、PCAGGS-VP2/4/7、PCAGGS-VP2/6/7共转染至293T细胞中,通过调整转染时间和转染方法,使用变性胶和非变性胶、透射电镜分别鉴定共转染后的结果,结果表明成功构建出PCAGGS-VP2/4/6、PCAGGS-VP2/6/7、PCAGGS-VP2/4/7病毒样颗粒,其粒子在形态、直径大小上都接近真实的病毒粒子。更多还原

【Abstract】 Porcine rotavirus（PoRV）belongs to the Reoviridae family,Rotavirus,in the etiological classification,and is a double-stranded RNA virus.Rotavirus infection of animals can make animals appear vomiting,diarrhea,dehydration,anorexia and other symptoms,because rotavirus infection is usually accompanied by other diarrhea virus co-infection or bacterial mixed infection,has caused greater economic losses in the world,in recent years,with the continuous expansion of China’s pig breeding industry,rotavirus to pig breeding industry losses are also expanding,in China’s northeast,northwest,north,central China,south and southwest and other regions,rotavirus incidence reached more than 40%,It shows that rotavirus is widely circulating in China.Since rotavirus currently has no specific drug,the treatment and prevention and control of adult pigs are relatively easy,but the treatment and prevention and control of piglets is more important,only safe and effective vaccination of piglets can reduce the morbidity and mortality of piglets and reduce the loss caused by rotavirus at the source.Virus-like particles are similar to the living virus itself in the form of proteins,but do not contain nucleic acid substances in live viruses,and because virus-like particles are formed by a variety of structural proteins,in theory,this multi-subunit protein is higher than that of a single subunit protein in animal organisms,and is similar to viruses in spatial morphology,so it has good immunogenicity and reactogenicity,foreign countries have successfully assembled VLPs research reports,and have made good progress in vaccine research.This provides a feasible idea for this experiment.This experiment studies and explores two types of rotavirus vaccines,and attempts to use the method of passaging weakening in traditional vaccines,which lays the foundation for the development of a new live attenuated rotavirus vaccine.In terms of new vaccines,attempts were made to assemble four important structural proteins of rotavirus on the eukaryotic cell platform,and finally constructed virus-like particles formed by different structural proteins,which laid the foundation for the subsequent research and development of rotavirus virus-like particle vaccines.The specific research is as follows:1.Passage mutation analysis of NJ2012Rotavirus can cause severe diarrhea in piglets,although the subunit vaccine or virus-like particle vaccine has a better immune effect,but the side effects of its immunization injection are relatively large compared to live attenuated vaccines,and in some cases may not be applicable,and China’s rotavirus live attenuated vaccine related research is relatively lacking,in order to study rotavirus live attenuated vaccine,the NJ2012 strain is continuously passaged to P56 generation,and the virus titer is detected every 10 generations during the passage process,NJ2012 P10/The virus titers of P20/P30/P40 reached 10^7.6TCID50/ml,10^7.8TCID50/ml,10^8.2TCID50/ml and 10^8.3TCID50/ml,respectively,and the results showed that the virus titer increased slowly with the increase of passages.The viral liquid of P30 and P40 was extracted from RNA and PCR amplification,and the VP2/VP4/VP6/VP7target bands with sizes of about 2700bp,2000bp,1500bp and 1000bp were successfully amplified,and the amplified products were sequenced by T vector ligation,and the sequencing results showed that P30 and P40 generations had point mutations,insertion mutations and deletion mutations on the four genes of VP2,VP4,VP6 and VP7.Point mutations trigger partial amino acid mutations,and there are no large fragments of nucleotide deletions or amino acid deletions.Using DNASTAR and NCBI,the whole gene sequences of VP2,VP4,VP6 and VP7 of several representative strains on GenBank were selected,and the gene sequences corresponding to P30 and P40 were compared with the gene sequences corresponding to P30 and P40,and it was found that the homology of the passaged NJ2012-P30/P40and other strains could reach between 70%-99.9%,among which the VP2 gene had the highest homology with the OSU strain,which was 99.5%.VP4 gene had the highest homology with OSU-C5111 strain,P30 and P40 had 83%and 95.4%homology with it,VP6 gene had high homology with K71,OSU and other strains,P30 and P40 had 95.2%and 95.5%homology with it,and VP7 gene had 99.9%homology with HBYC/2015/G9 strains.The amino acid homology of P30 and P40strains was lower than that of nucleotides,indicating that there were more amino acid changes during mutation.2.Construction of porcine rotavirus virus-like particlesIn order to explore whether the four structural proteins VP2/4/6/7 of the rotavirus strain NJ2012 can be successfully assembled to form virus-like particles,how to assemble,and what are the results after assembly,referring to the full-length gene of the rotavirus strain NJ2012 VP2/4/6/7 published in NCBI,primer design was carried out for these four genes,and four pairs of primers with enzyme cleavage sites and His’s tags were synthesized,using these four pairs of primers with NJ2012 as a template.The full-length gene of VP2/4/6/7 was successfully amplified.The amplified genes were cloned into PMD-19T vectors,and the positive recombinant plasmids were screened for sequencing,and the sequencing results showed that the nucleotide homology of the amplified genes and NJ2012 VP2/4/6/7 reached 99.7%,98.6%,99.8%and 99.9%,respectively,and the nucleotide mutations were all point mutations,and the resulting point mutations had no effect on the translated amino acids,and the required digestion sites and His tags were successfully amplified.After amplifying the target fragment,the four target genes were cloned to the polyclonal sites between EcoRI,Xho I,Kpn I and Xho I of the eukaryotic cell expression vector PCAGGS,and after PCR,single digestion and double digestion identification,the successfully constructed recombinant plasmid was transformed into trans5αcompetent cells,and four recombinant plasmids were expanded and cultured,named PCAGGS-VP2/4/6/7.After transfecting plasmids with 293T cells alone,the expression of four plasmids on 293T cells was successfully confirmed using Western-blot and indirect immunofluorescence.PCAGGS-VP2/4/6,PCAGGS-VP2/6,PCAGGS-VP2/4/7,PCAGGS-VP2/6/7 were co-transfected into 293T cells,and the results of co-transfection were identified by denaturing glue and non-denaturing glue and transmission electron microscopy by adjusting the transfection time and transfection method,and the results showed that PCAGGS-VP2/4/6 and PCAGGS-VP2/4/7 virus-like particles were successfully constructed.The diameter is close to that of real virions.更多还原