不同亚型兔出血症病毒单克隆抗体的制备及抗原表位鉴定

发布时间：2018-06-14 12:10

本文选题：兔出血症病毒 + 兔出血症病毒2型　；参考：《中国农业科学院》2015年硕士论文

【摘要】：兔出血症病毒(Rabbit Haemorrhagic Disease Virus,RHDV)隶属于嵌杯病毒科兔病毒属,是成年兔急性、烈性、高度接触性传染病,即兔病毒性出血症(Rabbit Haemorrhagic Disease,RHD)的病原体。2010年在法国的家兔和野兔中发现了一种新型的RHDV突变体,并命名为RHDV2。研究表明,RHDV1(传统的RHDV)和RHDV2有着不同的抗原性和遗传特性,系统进化树分析RHDV2为一个独立的分支,可能为兔病毒属的一个新的成员。在我国还没有对RHDV2的报道,加强对RHDV的病原学、流行病学、诊断方法和预防措施的综合性研究显得十分必要和紧迫。VP60蛋白是RHDV的主要衣壳蛋白,在机体诱导抗病毒感染的免疫反应中起着重要的作用,是RHDV的免疫保护性抗原。本实验首先设计引物扩增RHDV1 TP株(登录号:AF453761.1)的VP60序列,并克隆到原核表达载体p ET-32a中,构建重组表达载体p ET-R1-VP60。合成Gen Bank中已发表RHDV2 10-32株(登录号:HE800532.1)的VP60序列并将其克隆至原核表达载体p ET-32a中,构建重组表达载体p ET-R2-VP60。阳性质粒分别转化E.coli BL21 Star(DE3)p Lys S表达菌,经IPTG诱导后两种亚型的VP60重组蛋白p VP60-1和p VP60-2均得到大量表达。KCl染色后切胶纯化VP60重组蛋白,Western blot检测表达的p VP60-1和p VP60-2均具有良好的抗原性。将纯化的p VP60-1和p VP60-2分别与佐剂混合作为免疫原分别免疫BALB/c小鼠,通过杂交瘤融合技术将免疫后的小鼠脾细胞与SP2/0细胞进行融合。通过比对RHDV1和RHDV2的氨基酸序列,合成了6段差异性多肽。以p VP60-1和p VP60-2、合成的差异肽以及RHDV1 TP株作为检测抗原,建立间接ELISA检测方法筛选分泌抗RHDV1及抗RHDV2单克隆抗体(MAb)的杂交瘤细胞。通过有限稀释法进行3次亚克隆,共筛选到20株稳定分泌抗RHDV1的单抗株,其中有3株仅能识别RHDV1,分别命名为1D6、1H2、3F2;筛选到15株稳定分泌抗RHDV2的单抗株,其中有4株仅能识别RHDV2,分别命名为1G2、2C1、3B7、5D6。为了进一步鉴定这7株MAb的差异识别性,应用真核表达的VP60重组蛋白对7株MAb进行间接免疫荧光(IFA)和Western blot分析。首先采用体内诱生腹水法制备7株MAb的单克隆抗体。利用Bac-to-Bac昆虫杆状病毒表达系统,将表达RHDV1 vp60基因的重组杆状病毒(Bac-R-VP60)和表达RHDV2 vp60基因的重组杆状病毒(Bac-R2-VP60)接种Sf9细胞,分别表达了s VP60-1蛋白和s VP60-2蛋白,对7株MAb进行IFA鉴定。以真核表达载体pc DNA3.1(+)为骨架构建pc DNA-R1-VP60和pc DNA-R2-VP60重组质粒,转染hela细胞分别表达了RHDV1型VP60蛋白e VP60-1蛋白和RHDV2型e VP60-2蛋白,对7株MAb进行Western blot和IFA鉴定。IFA和Western blot结果表明1D6、1H2和3F2单独识别RHDV1 VP60蛋白,其中1D6的抗原表位为256RWNGQ260,1H2和3F2的抗原表位相同均为312VLQFW316;1G2、2C1、3B7和5D6单独识别RHDV2 VP60蛋白,其中2C1的抗原表位为324ADNPIS329,1G2、3B7和5D6的抗原表位相同均为294AIDHD298(倾斜字体为不同亚型中表达差异的氨基酸)。将鉴定的抗原表位与NCBI发布的兔病毒属成员VP60蛋白的氨基酸序列比对分析的结果表明只针对RHDV1 VP60蛋白单抗的抗原表位同源性为98%,而只针对RHDV2 VP60蛋白单抗的抗原表位同源性为100%。以上结果表明筛选得到的差异识别单抗的差异识别性比较稳定,能够区分不同亚型的RHDV,为型特异性单抗。本研究通过杂交瘤融合技术制备了RHDV病毒型特异性单克隆抗体,并鉴定出了特异性单抗的抗原表位。型特异性单抗株的制备为RHDV的鉴别诊断建立奠定了基础,对RHDV的流行病学调查,遗传进化分析及防控具有重要的意义。
[Abstract]:Rabbit Haemorrhagic Disease Virus (RHDV) belongs to the genus rabbit virus of the inlay Vivian family. It is an acute, intense, highly contagious disease of adult rabbits, that is, the pathogen of the Rabbit Haemorrhagic Disease (RHD) of rabbit (Rabbit Haemorrhagic Disease, RHD). A new RHDV mutant was found in the rabbit and rabbit in the French rabbit and hare. RHDV2. studies have shown that RHDV1 (traditional RHDV) and RHDV2 have different antigenicity and genetic characteristics. Phylogenetic tree analysis of RHDV2 is an independent branch and may be a new member of the rabbit virus. There is no report on RHDV2 in China to strengthen the synthesis of the pathogeny, epidemiology, diagnostic methods, and preventive measures of RHDV. It is necessary and urgent that.VP60 protein is the main capsid protein of RHDV, which plays an important role in the immune response of the organism to induce antiviral infection. It is the immune protective antigen of RHDV. First, primers were designed to amplify the VP60 sequence of the RHDV1 TP strain (login number: AF453761.1) and clone it to the prokaryotic expression vector p ET-32a. The recombinant expression vector p ET-R1-VP60. synthesized in Gen Bank has published the VP60 sequence of RHDV2 10-32 (login number: HE800532.1) and cloned it into the prokaryotic expression vector p ET-32a, and the recombinant expression vector was constructed, and P ET-R2-VP60. positive plasmids were transformed into two subtypes. The histone P VP60-1 and P VP60-2 all expressed a large number of.KCl stained VP60 recombinant proteins after.KCl staining. P VP60-1 and P VP60-2 detected by Western blot have good antigenicity. The mouse splenocytes were fused with the SP2/0 cells. By comparing the amino acid sequences of RHDV1 and RHDV2, 6 differential peptides were synthesized. The differential peptide and RHDV1 TP strain were used as detection antigens with P VP60-1 and P VP60-2, and an indirect ELISA detection method was established to screen hybridoma cells that secreted anti RHDV1 and anti RHDV2 monoclonal antibodies. The finite dilution method was used for 3 sub clones, and 20 stable secreting anti RHDV1 monoclonal antibodies were screened, of which 3 could only identify RHDV1, named 1D6,1H2,3F2, and 15 strains of anti RHDV2 monoclonal antibodies were screened, of which 4 were only able to identify RHDV2, respectively named 1G2,2C1,3B7,5D6. to identify the difference identification of the 7 strains of MAb further. The recombinant protein of eukaryotic expression VP60 was used to analyze 7 strains of MAb by indirect immunofluorescence (IFA) and Western blot. First, 7 MAb monoclonal antibodies were prepared by induced ascites in vivo. The Bac-to-Bac insect baculovirus expression system was used to express the heavy group of RHDV1 VP60 Gene baculovirus (Bac-R-VP60) and the expression RHDV2 VP60 Gene. The recombinant baculovirus (Bac-R2-VP60) was inoculated with Sf9 cells, and s VP60-1 protein and s VP60-2 protein were expressed respectively. The IFA of 7 strains of MAb was identified. The eukaryotic expression vector PC DNA3.1 (+) was used as the skeleton to construct PC DNA-R1-VP60 and recombinant plasmid. The results of Western blot and IFA identification of 7 MAb strains showed that 1D6,1H2 and 3F2 separately identified RHDV1 VP60 protein, and the antigen epitopes of 1D6 were the same as those of the antigen epitopes. The antigen epitopes of 7 and 5D6 are the same as 294AIDHD298 (inclined fonts are different amino acids in different subtypes). The results of the identification of antigen epitopes and the amino acid sequence of VP60 protein of the rabbit virus of NCBI show that the epitopes of the RHDV1 VP60 protein monoclonal antibody are only homologous to the antigen epitopes, but only for RHDV2 VP60. The antigen epitope homology of the protein monoclonal antibody was more than 100%.. The results showed that the differential identifiability of the screened monoclonal antibody was stable and could distinguish RHDV from different subtypes. The specific monoclonal antibody of RHDV virus type was prepared by hybridoma fusion technique, and the specific monoclonal antibody was identified. The preparation of the original epitope specific monoclonal antibody lay the foundation for the differential diagnosis of RHDV, and it is of great significance for the epidemiological investigation of RHDV, the analysis of genetic evolution and prevention and control.
【学位授予单位】：中国农业科学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.65

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