【作者】 马爱霞； 丁壮； 李娜； 宋子运； 徐明； 宣华；

【Author】 Ma aixia Ding Zhuang Li na Song ziyun Xu ming Xuan hua(College of Animal Science and Veterinary Medicine,Jilin University,Changchun 130062)

【机构】 吉林大学预防兽医学国家重点学科动物重要病原与疫病研究室；

【摘要】 将NA-1株鹅源副粘病毒毒种接种于11日龄SPF鸡胚,收集36～72h死亡的鸡胚尿囊液。参考已发表的鹅源副粘病毒M基因序列,设计并合成了两对特异性引物,用以扩增鹅源副粘病毒NA-1株F、HN基因。预期扩增的基因片段包含完整的开放阅读框。通过RT-PCR扩增出鹅源副粘病毒NA-1株F、HN基因片段。琼脂糖凝胶电泳回收、纯化,得到F、HN基因片段.经EcoR I,Sal I消化。将F、HN基因克隆进入pET-28a载体,转化大肠杆菌DH5a,挑选菌落。经限制性核酸内切酶Sal I和EcoR I双酶切及PCR鉴定,结果证明重组真核表达载体pET-F、pET-HN构建成功,将构建的表达载体分别转化表达菌BL21(DE3)中进行IPTG诱导表达。经SDS-PAGE分析,原核表达的F、HN蛋白分子量在59 Ku,63Ku,1 mM(终浓度)IPTG时间梯度诱导表达,3 h时F蛋白表达产量最高,占总菌体蛋白含量的23.9%;5 h时HN蛋白表达量最高,占整个菌体蛋白含量的26.2%;Western-blot检测发现原核表达的重组蛋白分别能与鼠抗GPMV阳性血清反应,证明原核表达的F蛋白、HN蛋白具有一定的反应原性。更多还原

【Abstract】 GPMV NA-1 was multiplied by SPF embryonated eggs and purified,and then to extract the RNA genome of virus. Based on the complete genome sequence of goose paramyxovirus strain NA-1 logged in GenBank and then two pairs specific primers were designed to amplified M,F and HN genes fragment by the method of RT-PCR.Meanwhile,the target gene fragments were respectively cloned into the pET-28a and were dentificated by restriction enzyme digestion and sequencing analysis.The positive recombinant plasmids were named pET-F,pET-HN after identification by restriction enzyme digestion.The prokaryotic expression vectors pET-F and pET-HN were constructed by inserting the F gene and HN gene into the pET-28a vector respectively.And the expression were induced by IPTG in E.coli B121(DE3).SDS-PAGE and Western-blot analysis showed that F protein amounted to 23.9 % of the total protein of E.coli BL21(DE3)after induced by IPTG(1 mmol/L)at 37℃for 3 hours,and HN protein amounted to 26.2% for 5 hours.The molecular weight of the expressed F protein and HN protein were about 59Ku,63Ku,and the proteins were specifically recognized by polyclonal antibody against GPMV.更多还原