猪流感病毒A/Swine/Inner Mongolia/547/01分离株神经氨酸酶基因的克隆及真核表达

Cloning and expressing in the baculovirus expression system of NA gene of A/Swine/Inner Mongolia/547/01 isolate

根据GenBank中登录的猪流感病毒参照序列设计了扩增神经氨酸酶 (NA)基因的特异性引物。从猪流感病毒A/Swine/InnerMongolia/5 4 7/0 1(H3N2 )株感染的鸡胚液中直接提取病毒RNA ,经RT_PCR扩增后将其克隆到pMD18_T载体上 ,进行序列测定和拼接。用HindⅢ酶切后 ,亚克隆到杆状病毒转移载体pMelBacB上 ,得到重组转移载体pMelBacNA ,然后与线性化杆状病毒DNA(Bac_N_BlueTMDNA)共转染于sf9昆虫细胞。经 3轮噬斑纯化 ,提取其DNA经PCR鉴定 ,获得重组杆状病毒株。SDS_PAGE结果显示 ,目的基因已获得表达 ,Westernblot和神经氨酸酶活性检测结果显示表达产物具有良好的免疫原性和神经氨酸酶活性 。

We designed a pair of primers to amplify the NA gene of Swine Influenza Virus (SIV) A/Swine/Inner Mongolia/547/01 (SW/IM/547/01) based on the reported gene sequence.The complimentary DNA of NA gene was amplified by RT-PCR and was cloned into pMD18-T vector.The NA gene was subcloned into the transfer vector pMelBacB after being digested with HindⅢ.Then the purified pMelBacNA was co-transfected to log-phase sf9 insect cells with the linear DNA of baculovirus (Bac-N-Blue^(TM) DNA) and Cellfectin reagent.The recombinant baculovirus were purified by three cycles of plaque assay with the chromogenic substrate x-gal in the agarose overlay.After that,the purified recombinant baculovirus was confirmed by PCR that it was not contaminated with field baculovirus.The results of SDS-PAGE indicated that NA gene was expressed successfully in sf9 insect cells.Western blot and NA assay indicated that the NA expressed by recombinant baculovirus in sf9 insect cells has good immunity and biological activity.The research is a good base for development of SIV subunit vaccine and the further study for the function of NA.