猪圆环病毒2型Cap蛋白的原核表达及其单克隆抗体制备  

Prokaryotic expression of Porcine circovirus type 2 Cap protein and preparation of its monoclonal antibody

作　　者：孙荣航 容芳 陈桂娥 刘郁夫 陈瑞爱 SUN Ronghang;RONG Fang;CHEN Guie;LIU Yufu;CHEN Ruiai(Zhaoqing Branch Center of Guangdong Laboratory for Lingnan Modern Agricultural Science and Technology,Zhaoqing 526238,China;College of Life Sciences,Zhaoqing University,Zhaoqing 526060,China;Huanong(Zhaoqing)Bioindustrial Technology Research Institute Co.,Ltd.,Zhaoqing 526238,China;College of Veterinary Medicine,South China Agricultural University,Guangzhou 510642,China;Zhaoqing Dahuannong Biological Medicine Co.,Ltd.,Zhaoqing 526238,China)

机构地区：[1]岭南现代农业科学与技术广东省实验室肇庆分中心,广东肇庆526238 [2]肇庆学院生命科学学院,广东肇庆526060 [3]华农(肇庆)生物产业技术研究院有限公司,广东肇庆526238 [4]华南农业大学兽医学院,广州510642 [5]肇庆大华农生物药品有限公司,广东肇庆526238

出　　处：《黑龙江畜牧兽医》2024年第14期61-67,73,126,共9页Heilongjiang Animal Science And veterinary Medicine

基　　金：肇庆市重点领域研发计划“揭榜制”项目(2021C001);岭南现代农业科学与技术广东省实验室肇庆分中心自主项目(P20211154-0201)。

摘　　要：为了制备猪圆环病毒2型(Porcine circovirus type 2,PCV-2)Cap蛋白单克隆抗体,试验将Cap基因克隆至原核表达载体pET-28a(+)上并转化至大肠杆菌BL21(DE3)感受态细胞中,将获得的重组表达菌pET28a-Cap/BL21(DE3)用IPTG进行诱导后表达重组蛋白,利用His标签对表达的重组蛋白进行纯化;将纯化后的重组蛋白于皮下免疫Balb/c小鼠,免疫结束后测定免疫小鼠血清效价,当小鼠血清抗体效价达到1∶100000时进行细胞融合;采用有限稀释法进行亚克隆,筛选能够稳定分泌抗体且抗体效价高的阳性杂交瘤细胞株,鉴定杂交瘤细胞株抗体亚类,并选择1株抗体效价最高的细胞株再次注射到小鼠腹腔中(5×10^(5)个/只),取腹水采用亲和层析法对单克隆抗体进行纯化;采用间接ELISA方法测定单克隆抗体效价并检测其特异性,分别采用Western-blot和间接免疫荧光试验检测单克隆抗体的反应性。结果表明:PCR扩增得到大小为708 bp的Cap基因,经双酶切与测序验证后,成功构建重组表达质粒pET28a-Cap;重组表达菌pET28a-Cap/BL21(DE3)经诱导后表达的重组蛋白可与His标签抗体发生特异性反应,纯化后的重组蛋白在预期位置(31.7 ku)出现清晰的单一条带;共筛选出8株能够稳定分泌抗体且抗体效价高的阳性单克隆细胞株(1E5、2A9、3C6、4B3、5F7、6D11、7A2、8G1株),抗体亚类鉴定均为IgG1亚类。其中7A2株抗体效价最高,用于单克隆抗体的制备;间接ELISA方法证实7A2株单克隆抗体仅与PCV-2 Cap蛋白发生反应,与猪繁殖与呼吸综合征病毒(Porcine reproductive and respiratory syndrome virus,PRRSV)、猪流行性腹泻病毒(Porcine epidemic diarrhea virus,PEDV)、猪瘟病毒(Classical swine fever virus,CSFV)、猪细小病毒(Porcine parvovirus,PPV)和猪伪狂犬病病毒(Porcine pseudorabies virus,PRV)不发生反应;Western-blot和间接免疫荧光试验均证实PCV-2 Cap蛋白可被7A2株单克隆抗体特异性识别。说明本试验成功In order to prepare monoclonal antibody against Cap protein of Porcine circovirus type 2(PCV-2),the Cap gene was cloned onto the prokaryotic expression vector pET-28a(+)and transformed into Escherichia coli BL21(DE3)receptor cells.Recombinant expression bacterium pET28a-Cap/BL21(DE3)was induced by IPTG to express the recombinant protein,and purified by His label.BALB/c mice were immunized subcutaneously with the purified recombinant protein,and the serum titer of the immunized mice was determined after the immunization,and cell fusion was performed when the serum antibody titer of the mice reached 1∶100000.Subcloning was performed by limited dilution method;positive hybridoma cell lines capable of stable antibody secretion and high antibody titer were screened;antibody subclasses of hybridoma cell lines were identified;a cell line with the highest antibody titer was selected to be injected into the abdominal cavity of mice again(5×10^(5)per cell),and ascites were taken for monoclonal antibody purification by affinity chromatography.The titer and specificity of monoclonal antibody were determined by indirect ELISA,and the reactivity of monoclonal antibody was detected by Western-blot and indirect immunofluorescence assay(IFA),respectively.The results showed that the Cap gene with the size of 708 bp was amplified by PCR,and the recombinant expression plasmid pET28a-Cap was successfully constructed after double digestion and sequencing.The recombinant protein expressed by the recombinant expression bacterium pET28a-Cap/BL21(DE3)after induction could react specifically with His labeled antibody,and the purified recombinant protein showed a clear single band at the expected location(31.7 ku).A total of 8 positive monoclonal cell lines(1E5,2A9,3C6,4B3,5F7,6D11,7A2,8G1)with high antibody titer and stable antibody secretion were screened,and the antibody subclass was identified as IgG1 subclass.Strain 7A2 had the highest titer and was used for the preparation of monoclonal antibody.Indirect ELISA confirmed that monocl

关 键 词：猪圆环病毒2型(PCV-2) 单克隆抗体 CAP蛋白 细胞融合 亚克隆 

分 类 号：S855.3[农业科学—临床兽医学]