禽源鹦鹉热衣原体MOMP基因重组腺病毒载体的构建与鉴定

Construction and identification of recombinant adenovirus with MOMP gene of Chlamydia psittaci of avian origin

对从疑似禽衣原体病鸡分离鉴定的鹦鹉热衣原体用PCR方法扩增,获得了完整的外膜主蛋白(MOMP)基因,将其克隆到pMD 18-T Simple载体中,用BglⅡ和SalⅠ双酶切出目的基因,并将其与已线性化的pshuttle-CMV穿梭载体连接。然后,将阳性质粒转化入含有腺病毒骨架载体的BJ5183感受态细胞中进行同源重组。将同源重组的腺病毒粒子转入DH5α大肠埃希氏菌增殖。提取同源重组后的腺病毒粒子转染AD-293细胞,待重组腺病毒在AD-293细胞中传代稳定后,经PCR技术检测,扩增到了MOMP目的基因;用间接免疫荧光试验检测,呈阳性反应,证明目的蛋白得到了表达。经测定,重组腺病毒的效价达3.9×1010PFU/mL。

A complete MOMP gene was amplified from Chlarnydia psittaci of chickens with avian chlamydiosis by PCR, and the amplicon was cloned correctly into a pMD 18-T Simple vector. The MOMP gene digested from the pMD 18-T Simple vector by BglⅡ and Sal I was linked with a linearized pshuttleCMV vector to obtain linearized positive plasmids. The linearized positive plasmids were transformed into BJ5183 competent cells containing a adenovirus framework vector for homologous recombination and the resultant recombinants were transformed into DH5a cells for further replication. The recombined adenovirus particles containing the MOMP gene were extracted and used to transfect AD-293 cells. When the recombinant adenovirus became adaptable to AD-293 cells following continuous passages,the target gene was detected by PCR and the expressed product was detected by indirect immunofluorescence test. Titers of the recombinant adenovirus was 3.9 X 10^10 PFU/mL.