猫传染性腹膜炎病毒N蛋白的原核表达及多抗制备

Prokaryotic Expression of N Protein of Feline Infectious Peritonitis Virus and Preparation of Polyclonal Antibodies

猫传染性腹膜炎病毒(FIPV)是冠状病毒中的重要成员,也是引起猫传染性腹膜炎的主要病原.参考GenBank数据库中FIPV核衣壳蛋白基因序列,设计特异性引物,以病料组织中提取的RNA为模板,反转录成cDNA,通过PCR特异性扩增N基因序列,将其克隆至pMD19-T载体并转化至Trans5α感受态细胞,经筛选、菌液PCR与限制酶双酶切鉴定后,获得重组克隆质粒pMD19-T-N.以pMD19-T-N为模板,亚克隆N基因,将其与Pet-28a载体连接后转化至表达菌株Rosetta感受态细胞,完成Pet-28a-N重组表达质粒的构建.用IPTG诱导N蛋白表达,将纯化后的重组N蛋白皮下多点注射免疫比利时兔,制备多克隆抗体.最终通过Western-Blot与免疫组化技术检测多克隆抗体的特异性.FIPV N蛋白多克隆抗体的制备,为猫传染性腹膜炎的进一步研究及临床治疗提供了必备的实验材料.

FIPV(feline infectious peritonitis virus),an important member of coronavirus,is the main cause of infectious peritonitis in cats.In this study,specific primers were designed by referring to the nucleocapsid protein gene N sequence of FIPV in GenBank database.Using the RNA extracted from diseased tissues as the template,the N gene sequence was amplified by RT-PCR,and cloned into pMD19-T vector and transformed into Trans5αcompetent cells.The recombinant plasmid pMD19-T-N was successfully constructed after antibiotics resistance gene screening,PCR and double enzyme digestion.Using pMD19-T-N as the template,N gene wassubcloned,and linked with Pet-28a vector and transformed into Rosetta competent cells to construct the Pet-28a-N recombinant expression plasmid.The expression of N protein was induced by IPTG,and polyclonal antibodies were prepared by subcutaneous immunization of Belgian rabbits with the purified N protein.Finally,the specificity of the polyclonal antibodies was detected by Western Blot and immunohistochemistry.The preparation of FIPV N protein polyclonal antibodies provides necessary experimental materials for further study and clinical treatment of infectious peritonitis in cats.