CAJ | 학술논문

为获得猪圆环病毒Ⅱ型 ORF3编码蛋白，研究该蛋白的特性及功能，根据 GenBank 数据库中PCVⅡ的基因组序列设计引物，利用 PCR 从病料基因组 DNA 中扩增 PCVⅡ河南地方株 ORF3，将其克隆至表达载体 pET 32a 中进行诱导表达，SDS PAGE 电泳检测重组蛋白表达情况，采用 Ni＋NTA 亲和纯化表达产物，透析复性后免疫日本大白兔制备多抗血清，并采用 ELISA 和 Western blot方法检测多抗血清的效价和特异性。结果显示，PCR 扩增获得全长315 bp 的 ORF3，重组质粒经测序和双酶切证实构建正确；SDS PAGE 结果显示，ORF3能够在大肠杆菌中表达，产物的分子量约为30 ku，以包涵体形式存在；纯化的重组蛋白具有较好的抗原性，制备的多抗血清抗体效价达1∶12800以上，抗体特异性高，能与表达的 ORF3编码蛋白结合。成功克隆了 PCVⅡ河南地方株 ORF3并实现原核表达，获得纯化的重组 ORF3编码蛋白。
위획득저원배병독Ⅱ형 ORF3편마단백，연구해단백적특성급공능，근거 GenBank 수거고중PCVⅡ적기인조서렬설계인물，이용 PCR 종병료기인조 DNA 중확증 PCVⅡ하남지방주 ORF3，장기극륭지표체재체 pET 32a 중진행유도표체，SDS PAGE 전영검측중조단백표체정황，채용 Ni＋NTA 친화순화표체산물，투석복성후면역일본대백토제비다항혈청，병채용 ELISA 화 Western blot방법검측다항혈청적효개화특이성。결과현시，PCR 확증획득전장315 bp 적 ORF3，중조질립경측서화쌍매절증실구건정학；SDS PAGE 결과현시，ORF3능구재대장간균중표체，산물적분자량약위30 ku，이포함체형식존재；순화적중조단백구유교호적항원성，제비적다항혈청항체효개체1∶12800이상，항체특이성고，능여표체적 ORF3편마단백결합。성공극륭료 PCVⅡ하남지방주 ORF3병실현원핵표체，획득순화적중조 ORF3편마단백。
The research aimed to obtain the recombinant ORF3 protein of porcine circovirus typeⅡ(PCVⅡ)to further research the characteristics and functions of ORF3 protein.According to the genomic sequence of PCV Ⅱ from GenBank,a pair of specific primers were designed.The ORF3 of Henan strain was amplified from pathological samples suspected PCVⅡ infection by PCR.Then the gene was cloned into prokaryotic expression vector pET-32a for expression in E. coli BL21(DE3).The expression product was purified by Ni+ NTA and refolded by dialysis.Japan white rabbits were immunized with the refolded recombinant ORF3 protein to generate polyclonal antiserum.The titer and specificity of the antiserum were detected respectively by ELISA and Western blot.The ORF3 of PCV Ⅱ,which was 315 bp in length,was amplified by PCR. Restriction enzyme digestion and sequencing proved that recombinant plasmid pET32a-ORF3 was constructed correctly.SDS-PAGE analysis showed that the ORF3 gene could express in E.coli BL21(DE3)and the expression prudct of ORF3 was about 30 ku,mainly in form of inclusion bodies.The purified recobinant ORF3 protein had good antigenicity.The specificty of polyclonal antiserum was high,and the titer could be above 1 ∶ 12 800.The ORF3 of PCV Ⅱ from Henan strain was successfully cloned and prokaryotic expression,which layed foundation for further research on the characteristics and functions of ORF3 protein.