摘要
利用overlap-PCR技术扩增获得鸡毒霉形体licA基因,将该基因经酶切克隆入原核表达载体pET32a(+)中,利用IPTG诱导进行原核表达,SDS-PAGE电泳结果显示,LicA呈现包涵体表达,利用His-Band Resin对包涵体进行纯化。将纯化产物作为免疫原腹腔注射BALB/c小鼠,制备获得LicA多克隆抗体。通过对鸡毒霉形体不同强弱毒株膜蛋白提取物进行Western-blot检测,发现在所有参试毒株中,LicA表达量恒定,并呈现膜定位特性。这一研究结果为进一步研究LicA生物学活性奠定了基础。
In this study,the licA gene of Mycoplasma gallisepticum was amplified by overlap PCR method,and then subcloned into a prokaryotic expression vector pET32a（＋） .The expression of LicA induced by IPTG was determined by SDS-PAGE analysis.After purification of the inclusion bodies,the His-LicA peptides were used as antigens to prepare the polyclonal antibodies against LicA.By western-blot analysis on the cell extracts of membrane proteins in all the virulent and attenuated strains,LicA showed a constant expression amount and localized on the cell membrane.The study on the biological activity of LicA would be carried out in future.
出处
《中国兽医科学》
CAS
CSCD
北大核心
2010年第11期1142-1145,共4页
Chinese Veterinary Science
基金
国家自然科学基金资助项目(30871883)
中央科研院所基础业务经费项目(2008JB12)
上海市科技兴农重点攻关项目(2007-11-2)
关键词
鸡毒支原体
licA基因
原核表达
膜定位
Mycoplasma gallisepticum
licA gene
prokaryotic expression
membrane localization
