本文選題：兔出血癥病毒 + 兔出血癥病毒2型��； 參考：《中國農(nóng)業(yè)科學(xué)院》2015年碩士論文

【摘要】：兔出血癥病毒(Rabbit Haemorrhagic Disease Virus,RHDV)隸屬于嵌杯病毒科兔病毒屬,是成年兔急性、烈性、高度接觸性傳染病,即兔病毒性出血癥(Rabbit Haemorrhagic Disease,RHD)的病原體。2010年在法國的家兔和野兔中發(fā)現(xiàn)了一種新型的RHDV突變體,并命名為RHDV2。研究表明,RHDV1(傳統(tǒng)的RHDV)和RHDV2有著不同的抗原性和遺傳特性,系統(tǒng)進(jìn)化樹分析RHDV2為一個獨立的分支,可能為兔病毒屬的一個新的成員。在我國還沒有對RHDV2的報道,加強對RHDV的病原學(xué)、流行病學(xué)、診斷方法和預(yù)防措施的綜合性研究顯得十分必要和緊迫。VP60蛋白是RHDV的主要衣殼蛋白,在機體誘導(dǎo)抗病毒感染的免疫反應(yīng)中起著重要的作用,是RHDV的免疫保護(hù)性抗原。本實驗首先設(shè)計引物擴增RHDV1 TP株(登錄號:AF453761.1)的VP60序列,并克隆到原核表達(dá)載體p ET-32a中,構(gòu)建重組表達(dá)載體p ET-R1-VP60。合成Gen Bank中已發(fā)表RHDV2 10-32株(登錄號:HE800532.1)的VP60序列并將其克隆至原核表達(dá)載體p ET-32a中,構(gòu)建重組表達(dá)載體p ET-R2-VP60。陽性質(zhì)粒分別轉(zhuǎn)化E.coli BL21 Star(DE3)p Lys S表達(dá)菌,經(jīng)IPTG誘導(dǎo)后兩種亞型的VP60重組蛋白p VP60-1和p VP60-2均得到大量表達(dá)。KCl染色后切膠純化VP60重組蛋白,Western blot檢測表達(dá)的p VP60-1和p VP60-2均具有良好的抗原性。將純化的p VP60-1和p VP60-2分別與佐劑混合作為免疫原分別免疫BALB/c小鼠,通過雜交瘤融合技術(shù)將免疫后的小鼠脾細(xì)胞與SP2/0細(xì)胞進(jìn)行融合。通過比對RHDV1和RHDV2的氨基酸序列,合成了6段差異性多肽。以p VP60-1和p VP60-2、合成的差異肽以及RHDV1 TP株作為檢測抗原,建立間接ELISA檢測方法篩選分泌抗RHDV1及抗RHDV2單克隆抗體(MAb)的雜交瘤細(xì)胞。通過有限稀釋法進(jìn)行3次亞克隆,共篩選到20株穩(wěn)定分泌抗RHDV1的單抗株,其中有3株僅能識別RHDV1,分別命名為1D6、1H2、3F2;篩選到15株穩(wěn)定分泌抗RHDV2的單抗株,其中有4株僅能識別RHDV2,分別命名為1G2、2C1、3B7、5D6。為了進(jìn)一步鑒定這7株MAb的差異識別性,應(yīng)用真核表達(dá)的VP60重組蛋白對7株MAb進(jìn)行間接免疫熒光(IFA)和Western blot分析。首先采用體內(nèi)誘生腹水法制備7株MAb的單克隆抗體。利用Bac-to-Bac昆蟲桿狀病毒表達(dá)系統(tǒng),將表達(dá)RHDV1 vp60基因的重組桿狀病毒(Bac-R-VP60)和表達(dá)RHDV2 vp60基因的重組桿狀病毒(Bac-R2-VP60)接種Sf9細(xì)胞,分別表達(dá)了s VP60-1蛋白和s VP60-2蛋白,對7株MAb進(jìn)行IFA鑒定。以真核表達(dá)載體pc DNA3.1(+)為骨架構(gòu)建pc DNA-R1-VP60和pc DNA-R2-VP60重組質(zhì)粒,轉(zhuǎn)染hela細(xì)胞分別表達(dá)了RHDV1型VP60蛋白e VP60-1蛋白和RHDV2型e VP60-2蛋白,對7株MAb進(jìn)行Western blot和IFA鑒定。IFA和Western blot結(jié)果表明1D6、1H2和3F2單獨識別RHDV1 VP60蛋白,其中1D6的抗原表位為256RWNGQ260,1H2和3F2的抗原表位相同均為312VLQFW316;1G2、2C1、3B7和5D6單獨識別RHDV2 VP60蛋白,其中2C1的抗原表位為324ADNPIS329,1G2、3B7和5D6的抗原表位相同均為294AIDHD298(傾斜字體為不同亞型中表達(dá)差異的氨基酸)。將鑒定的抗原表位與NCBI發(fā)布的兔病毒屬成員VP60蛋白的氨基酸序列比對分析的結(jié)果表明只針對RHDV1 VP60蛋白單抗的抗原表位同源性為98%,而只針對RHDV2 VP60蛋白單抗的抗原表位同源性為100%。以上結(jié)果表明篩選得到的差異識別單抗的差異識別性比較穩(wěn)定,能夠區(qū)分不同亞型的RHDV,為型特異性單抗。本研究通過雜交瘤融合技術(shù)制備了RHDV病毒型特異性單克隆抗體,并鑒定出了特異性單抗的抗原表位。型特異性單抗株的制備為RHDV的鑒別診斷建立奠定了基礎(chǔ),對RHDV的流行病學(xué)調(diào)查,遺傳進(jìn)化分析及防控具有重要的意義。
[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.
【學(xué)位授予單位】：中國農(nóng)業(yè)科學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S852.65

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本文編號：2017330