Analysis of peblA gene sequence and construction of eukaryotic expression recombinant plasmid of peblA gene of Campylobacter jejuni Pen:19 / 第三军医大学学报

ABSTRACT

Objective As the genome sequence of Campylobacter jejuni is a hypervariable sequence, the peblA gene sequence of Campylobacter jejuni Pen∶19 was sequenced and the peblA gene conservative was identified. To construct the eukaryotic expression recombinant plasmid of the peblA gene of Campylobacter jejuni Pen∶19 . Methods Total DNA of Campylobacter jejuni Pen∶2, Pen∶8, Pen∶19 and Pen∶21 as templates respectively, peblA gene DNA with KpnⅠ and EcoRⅠ sites was amplified by PCR using the same primer and the PCR product of Pen∶19 was sequenced. Because most close association with Guillain-Barre syndrome, the PCR product of Pen∶19 was selected as target gene and cloned into pcDNA3.1(+) and constructed the recombinant plasmid that was identified by endonuclease digestion and PCR and confirmed by sequencing. The recombinant plasmid was then transfected into mammalian cell COS-7 for expression. The transient expression was investigated by RT-PCR. The expression of peblA gene in the culture supernatant of positive clones was analyzed by ELISA. Results The peblA gene sequence of Campylobacter jejuni Pen∶2 and Pen∶19 was identical. The target gene was amplified from COS-7 cells transfected with recombinant plasmids by RT-PCR. PEBl protein could express in the culture supernatant in the transfected COS-7 cells by ELISA. Conclusion The peblA gene of Campylobacter jejuni was conservative. The recombinant plasmid was constructed and expressed in COS-7 cells successfully. The results obtained lay the foundation for research on development of Campylobacter jejuni DNA vaccine.