山羊副流感病毒3型HN基因原核表达与单克隆抗体的制备

发布时间：2018-09-10 12:18

【摘要】：副流感病毒3型(parainfluenza virus type 3,PIV3)属于副粘病毒科呼吸道病毒属,是囊膜的单股负链RNA病毒,是引起人和动物呼吸道疾病的重要病原,包括:人副流感病毒3型(Human parainfluenza virus type 3,HPIV3)和牛副流感病毒3型(Bovine parainfluenza virus type 3,BPIV3)。目前国内外对HPIV3与BPIV3的研究报道较为广泛,但PIV3在羊群中的感染情况在国内外都鲜有报道。本实验室从发病山羊中分离出一株新的PIV3病毒,命名为为山羊副流感病毒3型(Caprine parainfluenza virus 3,CPIV3)JS2013。因此为监测该病在羊群中的流行情况,急需建立一种病原学及血清学检测方法,为开展流行病学调查提供依据。PIV3基因组至少编码6种结构蛋白,即NP、P、M、F、HN和L蛋白,其中HN蛋白和F蛋白是副流感病毒3型的主要保护性抗原。HN蛋白存在至少6个抗原表位,其N端具有强烈的疏水性,从而使HN蛋白能够牢固地嵌入病毒囊膜的双层质膜内,3个中和性抗原表位均存在于其中。因此本研究利用大肠杆菌原核表达系统,成功表达PIV3 JS2013重组蛋白His-HN,并制备出针对CPIV3 HN的单克隆抗体,以期为副流感病毒3型诊断方法的建立和HN蛋白的结构和功能的研究奠定基础。主要研究内容包括:1、CPIV3 JS2013 HN基因片段克隆与表达参考Gen Bank中发表的牛副流感病毒3型(BPIV3)内蒙09毒株(NM09)全基因组序列(Gen Bank登录号:JQ063064),设计一对特异性引物扩增山羊副流感病毒3型(CPIV3)JS2013株HN基因部分序列,将扩增得到的基因进行测序,比对分析其与已报道HPIV3、BPIV3的序列同源性最高分别为74.3%、79.8%。进化分析表明JS2013株并不属于HPIV3与BPIV3,形成一个独立的分支。将HN基因片段克隆至原核表达载体p ET-32a(+),获得原核重组质粒p ET-32a-HN。将重组质粒转化到大肠杆菌BL21(DE3)感受态菌株中,经IPTG诱导表达,通过SDS-PAGE与His抗体western blot试验分析表明,重组蛋白His-HN诱导表达成功,并以包涵体形式存在,大小约为46 ku。Western blot试验验证重组蛋白免疫BALB/c小鼠制备的高免血清能够与重组蛋白反应;病毒接种MDBK细胞后,间接免疫荧光(IFA)试验表明其多克隆抗体与病毒产生特异性荧光反应,表明该重组蛋白具有很好的免疫原性和反应原性。2、CPIV3 JS2013 HN基因单克隆抗体的制备与鉴定用纯化后的重组蛋白免疫BALB/c小鼠,制备鼠抗His-HN蛋白的单克隆抗体。按照常规方法制备SP2/0骨髓瘤细胞和小鼠脾细胞进行融合,用间接ELISA筛选阳性克隆,采用有限稀释法进行亚克隆,经过3次亚克隆后筛选出3株能够稳定分泌抗His-HN蛋白的单克隆抗体,将其分别命名为1F8、2D5、4A7。其细胞培养上清和小鼠腹水ELISA效价分别为29、28、28和106、106、105。这三株杂交瘤细胞分泌的单克隆抗体的Ig亚型均为Ig G2b,轻链类型为κ。Western blot试验验证三株单克隆抗体与His-HN蛋白产生特异性反应;IFA与Dot-ELISA试验表明三株单克隆抗体均能够特异性识别CPIV3 JS2013株。三株单抗的研制成功为建立敏感性高、特异性强的CPIV3抗原诊断方法打下了基础。
[Abstract]:Parainfluenza virus type 3 (PIV3) belongs to the genus Paramyxovirus of the family Paramyxoviridae. It is a single-stranded negative-stranded RNA virus of the capsule. It is an important pathogen causing respiratory diseases in humans and animals, including human parainfluenza virus type 3 (HPIV3) and bovine parainfluenza virus type 3 (Bovine parainfluenza virus type 3). HPIV3 and BPIV3 have been extensively studied at home and abroad, but the infection of PIV3 in goats is rarely reported at home and abroad. A new PIV3 virus named Caprine parainfluenza virus 3 (CPIV3) JS2013 was isolated from infected goats in our laboratory. In order to provide evidence for epidemiological investigation, it is urgent to establish an etiological and serological method for the detection of the disease in sheep. The PIV3 genome encodes at least six structural proteins, namely, NP, P, M, F, HN and L proteins. HN and F proteins are the main protective antigens of parainfluenza virus type 3. HN proteins have at least six epitopes. The N-terminal of the recombinant protein was strongly hydrophobic, so that the HN protein could be firmly embedded in the bilayer plasma membrane of the virus envelope. Three neutralizing antigen epitopes were found in the bilayer plasma membrane. The main research contents include: 1. Cloning and expression of CPIV3 JS2013 HN gene fragment reference Gen Bank published bovine parainfluenza virus type 3 (BPIV3) Inner Mongolia 09 strain (NM09) whole genome sequence (Gen Bank login number: JQ063064), design a pair of specificity. The partial sequence of HN gene of JS2013 strain of goat parainfluenza virus type 3 (CPIV3) was amplified by primers. The amplified HN gene was sequenced and compared with HPIV3 and BPIV3. The highest sequence homology was 74.3% and 79.8% respectively. Evolutionary analysis showed that JS2013 strain did not belong to HPIV3 and BPIV3, forming an independent branch. The recombinant plasmid P ET-32a-HN was obtained from the prokaryotic expression vector p ET-32a (+). The recombinant plasmid was transformed into E. coli BL21 (DE3) susceptible strain and expressed by IPTG. SDS-PAGE and Western blot analysis showed that the recombinant protein His-HN was successfully induced and existed as an inclusion body, about 46 ku.Wester. The immunogenicity of the recombinant protein against BALB/c mice was confirmed by n-blot assay. The indirect immunofluorescence assay (IFA) showed that the polyclonal antibody of the recombinant protein could react with the recombinant protein. 2. CPIV3 JS2013 HN To prepare and identify monoclonal antibodies, BALB/c mice were immunized with purified recombinant protein to prepare monoclonal antibodies against his-HN protein. SP2/0 myeloma cells and mouse spleen cells were fused by conventional methods. Positive clones were screened by indirect ELISA, subcloned by limited dilution method, and screened after three subclones. Three monoclonal antibodies secreting His-HN protein stably were identified as 1F8, 2D5 and 4A7. The ELISA titers of the supernatants and ascites of mice were 29, 28, 28 and 106, 106, 105, respectively. The Ig subtypes of the monoclonal antibodies secreted by the three hybridoma cells were all Ig G2b, and the light chain type was kappa. Western blot test confirmed that the three monoclonal antibodies were monoclonal antibodies. The results of IFA and Dot-ELISA showed that all three monoclonal antibodies could specifically recognize CPIV3 JS2013 strains. The successful preparation of three monoclonal antibodies laid a foundation for the establishment of a highly sensitive and specific diagnostic method for CPIV3 antigen.
【学位授予单位】：安徽农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.65

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