藏鸡源乳酸菌的筛选鉴定及IBVN基因的表达研究

Isolation and identification of lacticacid bacteria from Tibetan chicken and expression of foreign protein in it

从藏鸡(海拔3000米藏区、自然放养、未使用过抗生素)肠道内筛选乳杆菌,经16s rDNA和生理生化试验鉴定为植物乳杆菌.通过耐酸耐胆汁试验和肠道黏附试验测试,表明其具有良好的耐酸耐胆汁能力和肠道黏附能力.为使外源质粒顺利转化入乳杆菌,进行电场强度的优化,再按照优化后的电场强度,将禽冠状病毒pSIP409-N重组质粒电转化入该株乳杆菌,进行双酶切和PCR鉴定.将经鉴定成功转入的重组乳杆菌,用诱导肽SppIP诱导表达,再进行SDS-PAGE检测外源蛋白的表达.结果表明N蛋白能够顺利表达.上述试验证明该株植物乳杆菌具有作为乳酸菌活载体疫苗的潜力.

One lactobacillus strain was isolated from free-ranging Tibetan chicken in Ganzi autonomous county, Sichuan Province. The strain was identified as Lactobacillus plantarum by 16s rDNA sequence and physiological and biochemical identification. The tests for growth with bile salts, tolerance to acidic pH, and adhesion efficiency to the epithelial cells of chickens were also carried out. These data sugges- ted the strain has a good ability to survive in gastrointestinal tract and a high adhesion efficiency to the epithelial cells. We then estimated the isolated lactobacillus as a host to express foreign genes. Recombi- nant plasmid pSIP409-N inserted nucleoprotein （N） of IB virus was electroporated into the strain, and the N protein was successfully expressed while it was induced by SppIP. These results indicated that the foreign recombinant plasmid can be transformed into L. plantarum and expressed efficiently. Conclusive- ly, the present study demonstrated that L. plantarurn is a potential live vector strain for poultry vaccine.