东北农业大学学报
東北農業大學學報
동북농업대학학보
JOURNAL OF NORTHEAST AGRICULTURAL UNIVERSITY
2014年
10期
57-63
,共7页
李广兴%杜威威%韩溪%潘龙%王新%白妍%洪琴%马玲%任晓峰%杨贵君
李廣興%杜威威%韓溪%潘龍%王新%白妍%洪琴%馬玲%任曉峰%楊貴君
리엄흥%두위위%한계%반룡%왕신%백연%홍금%마령%임효봉%양귀군
传染性支气管病毒%膜蛋白%多克隆抗体%间接免疫荧光试验%免疫印迹试验
傳染性支氣管病毒%膜蛋白%多剋隆抗體%間接免疫熒光試驗%免疫印跡試驗
전염성지기관병독%막단백%다극륭항체%간접면역형광시험%면역인적시험
IBV%membrane protein%polyclonal antibody%indirect fluorescence antibody assay%Western blot
文章通过RT-PCR方法扩增传染性支气管炎病毒（IBV）HH06株M全长基因，经抗原性和亲水性分析，将M部分序列成功亚克隆于pET-30a（+）和PVAX1载体中。将阳性重组质粒pET30a-M转化E. coli Rosetta （DE3）感受态细胞，诱导表达获得20 ku重组M蛋白。Western blot检测表明，重组蛋白能够与IBV参考阳性血清反应。以纯化重组M蛋白免疫新西兰白兔制备多克隆抗体，其ELISA效价达到1??218；Western blot结果证实，其具有良好的反应性和特异性。间接免疫荧光试验表明，抗M蛋白多克隆抗体可检测到PVAX-M1转染BHK-21细胞表达的M蛋白，与IBV HH06毒株具有很好的特异性反应。病毒感染抑制试验表明，多抗血清对IBV Beaudette株的抑制率可达到25.9%。为IBV检测及M蛋白功能的研究奠定基础。
文章通過RT-PCR方法擴增傳染性支氣管炎病毒（IBV）HH06株M全長基因，經抗原性和親水性分析，將M部分序列成功亞剋隆于pET-30a（+）和PVAX1載體中。將暘性重組質粒pET30a-M轉化E. coli Rosetta （DE3）感受態細胞，誘導錶達穫得20 ku重組M蛋白。Western blot檢測錶明，重組蛋白能夠與IBV參攷暘性血清反應。以純化重組M蛋白免疫新西蘭白兔製備多剋隆抗體，其ELISA效價達到1??218；Western blot結果證實，其具有良好的反應性和特異性。間接免疫熒光試驗錶明，抗M蛋白多剋隆抗體可檢測到PVAX-M1轉染BHK-21細胞錶達的M蛋白，與IBV HH06毒株具有很好的特異性反應。病毒感染抑製試驗錶明，多抗血清對IBV Beaudette株的抑製率可達到25.9%。為IBV檢測及M蛋白功能的研究奠定基礎。
문장통과RT-PCR방법확증전염성지기관염병독（IBV）HH06주M전장기인，경항원성화친수성분석，장M부분서렬성공아극륭우pET-30a（+）화PVAX1재체중。장양성중조질립pET30a-M전화E. coli Rosetta （DE3）감수태세포，유도표체획득20 ku중조M단백。Western blot검측표명，중조단백능구여IBV삼고양성혈청반응。이순화중조M단백면역신서란백토제비다극륭항체，기ELISA효개체도1??218；Western blot결과증실，기구유량호적반응성화특이성。간접면역형광시험표명，항M단백다극륭항체가검측도PVAX-M1전염BHK-21세포표체적M단백，여IBV HH06독주구유흔호적특이성반응。병독감염억제시험표명，다항혈청대IBV Beaudette주적억제솔가체도25.9%。위IBV검측급M단백공능적연구전정기출。
In this study, the complete IBV HH06 M gene was cloned and its partial gene was subcloned into prokaryotic expression vector pET-30a (+) and eukaryotic expression vector PVAX1 according to hydrophilicity plot and antigenic index analysis results. The recombinant plasmid of pET30a-M was transformed into E. coli Rosetta (DE3) and induced with IPTG, the molecular weight of recombinant M protein is 20 ku. The recombinant M protein could be recognized by positive IBV antisera in Western blot. Then the purified recombinant M protein was used as antigen for immunization of rabbit to prepare anti-M polyclonal antibody. Indirect ELISA showed that the titer of anti-M polyclonal antibody was 1??218, and it had highly reactivity and specialty in Western blot. Furthermore, IFA test demonstrated that this polyclonal antibody could react with BHK-21 cells transfected with PVAX-M1 plasmid and IBV-infected Vero cells. The pathogenic effect of Vero cells infected with IBV Beaudette strain could be inhibited by anti-M polyclonal antibody and the inhibition rate was up to 25. 9%. The rabbit anti-M protein polyclonal antibody obtained in this study laid a foundation for further functional research for M protein and detection of IBV.
