四川猪流行性腹泻病毒流行株S基因的分子特征与S蛋白抗原性的初步分析

发布时间：2018-05-12 23:14

本文选题：PEDV + S基因　；参考：《四川农业大学》2015年硕士论文

【摘要】：猪流行性腹泻(Porcine Epidemic Diarrhea, PED)是由猪流行性腹泻病毒(Porcine Epidemic Diarrhea Virus, PEDV)引起的一种高度接触性肠道传染疾病。自2010年以来,PEDV感染已成为全球养猪业共同关注的问题。及时掌握PEDV流行毒株的分子特征,对指导该病具有重要意义。S基因编码的S蛋白是PEDV的重要结构蛋白,在诱导机体产生中和抗体和提供免疫保护等方面起关键作用,S基因较易发生变异。本研究对四川2014年PEDV流行毒株的S基因(包括S基因全序列和部分序列)进行克隆、测序和分子特性分析,初步分析了流行株的S基因抗原性(包含COE区的抗原区域)。研究内容如下：(1)四川PEDV流行株S基因的克隆与分子特征分析：以RT-PCR检测了四川14个地区的17个猪场的102份发病仔猪腹泻样本,猪场PEDV阳性率达到100%(17/17),样品PEDV阳性率为88.2%(99/102)。PEDV多为单独感染。根据PEDV-CV777株序列(GenBank登录号：AF353511)设计3对引物扩增完整的S基因,选取2014年四川地区PEDV代表样品扩增了9株S基因全序列和18株S1区(起止位点：1-1389nt),采用RT-PCR扩增S基因、克隆、测序和序列分析。结果表明：S基因S1片段变异较大,18株流行株S1片段全长为1398bp,编码466个氨基酸,存在大量核苷酸的突变、插入及缺失,多以C→T、T→C、 G→T、T→G等突变形式存在。9株完整S基因中,8株全长为4161bp,编码1386个氨基酸,1株为4125bp,编码1374个氨基酸。PEDV四川流行株之间核苷酸序列同源性及氨基酸序列同源性均较高,与SC-L株及CV777疫苗株等的同源性均较低,与2013-2014年US株、Thailand、2014年韩国株等的同源性较高。S基因编码的多肽链具有较好的亲水性,分别含有1个跨膜区域及信号肽切割位点,潜在的磷酸化位点及糖基化位点与CV777差异较大。密码子偏嗜性分析表明S基因对编码的T'rp, Met, His, Asp, Cys氨基酸具有一定的偏嗜性。S蛋白含有多个α螺旋区段、β折叠区段、β转角区域及无规则卷曲区域。遗传进化分析结果表明,本研究中的PEDV四川地区流行毒株均属于同一群,18株四川流行株S1区与中国株CH8、YS、CHGD-01、GDMZ/2013、韩国株CNU-091222-01、美国株USA/Iowa/18984/2013、USA IA2、 USA/Indiana/17846/2013、USA/Kansas125/2014、US A/Texas 128/2014、最新韩国株Korea KUIDL-PED-2014-002及KUIDL-PED-2014-007、泰国株Thailand PPED0108、Thailand 1-55ST0412、Thailand 6-56ST0413、Thailand SBPED0211-2等亲缘关系较近。COE区与S全基因与参考株的亲缘关系与S1区相似,但COE区具有地区性差异,这提示四川境内流行的毒株也在不断进化。此外,研究中所有四川地区PEDV流行毒株均与经典毒株CV777、Brl/87,中国早期流行毒株LZC、SC-L、DX、韩国株Chinju99、DR13及日本株83p-5等亲缘关系较远。(2)S蛋白抗原性的初步分析：本研究设计了包含COE区的部分S蛋白片段(命名为SD蛋白)的特异性引物SD-F、SD-R,用RT-PCR技术扩增了PEDV四川乐山流行株及CV777疫苗株的892bp的抗原表位片段,并插入原核表达载体pET39b(+)中,分别构建了重组表达质粒pET39b-SC-SD及pET39b-Vaccine-SD。将重组表达质粒转化表达宿主菌BL21(DE3)中,经37℃,lmmol/L IPTG诱导表达4h, SDS-PAGE显示目的基因片段得到表达,目的蛋白分子量约为62kD,表达量较低。将包涵体切胶纯化,以获得的S蛋白抗原免疫家兔获得兔抗多克隆抗体,通过琼脂扩散法测定抗体效价,流行株及疫苗株的效价分别为1：8、1：4,同时对纯化的蛋白进行Western blot检测,表明获得的抗体对原核表达的SD蛋白具有特异性反应。
[Abstract]:Porcine Epidemic Diarrhea (PED) is a highly contagious intestinal infectious disease caused by the swine epidemic diarrhea virus (Porcine Epidemic Diarrhea Virus, PEDV). Since 2010, PEDV infection has become a common concern in the global pig raising industry. The S protein encoded by the.S gene is an important structural protein of PEDV, which plays a key role in inducing the body to neutralize antibodies and provide immune protection. The S gene is more susceptible to mutation. This study cloned the S gene of the PEDV epidemic strain of Sichuan in 2014, including the full and partial sequences of the S gene, and the sequence and molecule of the gene. The antigenicity of the S gene (including the antigen region of the COE region) was preliminarily analyzed. The contents of the study were as follows: (1) the cloning and molecular characterization of the S gene of Sichuan PEDV epidemic strain: 102 piglet diarrhoea samples were detected by RT-PCR in 17 pig farms in 14 regions of the City, and the positive rate of PEDV in the pig farm was 100% (17/17), and the sample PEDV The positive rate was 88.2% (99/102).PEDV as a single infection. According to the sequence of PEDV-CV777 strains (GenBank login number: AF353511), 3 pairs of primers were designed to amplify the complete S gene, and 9 S gene sequences and 18 S1 region (1-1389nt) were amplified by PEDV representative samples in 2014, and RT-PCR amplification of S genes, cloning, sequencing and sequence were used. The results showed that the S1 fragment of the S gene was highly variable. The total length of the 18 strains of the 18 strains was 1398bp, encoding 466 amino acids, and there were a large number of mutations, insertion and deletion of nucleotides, and there were more C to T, T to C, G to T, T to G, and the 8 strains were encoded 1386 amino acids, 1 strains were encoded 1374. The nucleotide sequence homology and amino acid sequence homology of the.PEDV Sichuan epidemic strains were high, and the homology of the SC-L strain and the CV777 vaccine strain was lower. The polypeptide chain with high homologous.S gene encoded by the 2013-2014 year US strain, Thailand, and 2014 Korean strain had better hydrophilicity, which contained 1 transmembrane regions, respectively. The potential phosphorylation sites and glycosylation sites are different from the CV777. The codon bias analysis shows that the S gene has a certain partial tropism of the encoded T'rp, Met, His, Asp, and Cys amino acids with a number of alpha helix segments, beta fold regions, beta corner regions and irregular curly regions. Genetic evolution points. The results show that all the epidemic strains in the PEDV Sichuan area belong to the same group, 18 strains of Sichuan epidemic strain S1 area and Chinese strain CH8, YS, CHGD-01, GDMZ/2013, Korean strain CNU-091222-01, American strain USA/Iowa/18984/2013, USA IA2, USA/Indiana/17846/2013, USA/. 002 and KUIDL-PED-2014-007, Thailand PPED0108, Thailand 1-55ST0412, Thailand 1-55ST0412, Thailand 6-56ST0413, Thailand SBPED0211-2 and so on, the relationship between the close.COE region and the S whole gene and the reference strain is similar to that of the S1 region, but the regional differences are regional, which suggests that the epidemic strains in Sichuan are also evolving. All the PEDV epidemic strains in Sichuan area were closely related to the classical strains CV777, Brl/87, Chinese early epidemic strains LZC, SC-L, DX, Korean strain Chinju99, DR13 and Japanese strain 83p-5. (2) preliminary analysis of the antigenicity of S protein: This study designed specific primers containing partial S protein fragments containing COE region. The epitope fragment of 892bp in Leshan Sichuan Leshan epidemic strain and CV777 vaccine strain was amplified by RT-PCR technique and inserted into the prokaryotic expression vector pET39b (+). The recombinant expression plasmid pET39b-SC-SD and pET39b-Vaccine-SD. were constructed to express the recombinant expression plasmid in BL21 (DE3), and the expression 4H was induced by 37 C and lmmol/L IPTG. AGE showed that the target gene fragment was expressed, the molecular weight of the target protein was about 62kD and the expression was low. The inclusion body was purified and the S protein antigen was purified to obtain the Rabbit anti polyclonal antibody and the antibody titer was measured by the agar diffusion method. The effective price of the epidemic strain and the vaccine strain was 1:8,1:4 respectively, and the purified protein was also carried out. Western blot showed that the antibody was specific for prokaryotic expression of SD protein.

【学位授予单位】：四川农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.65

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