表达副鸡禽杆菌外膜蛋白的重组乳酸乳球菌的构建及其免疫原性研究

Construction and identification of recombinant Lactococcus lactis expressing outer membrane protein of Avibacterium paragallinarum

为构建表达副鸡禽杆菌外膜蛋白的重组乳酸乳球菌,本试验按照乳酸乳球菌表达的偏好性,优化合成副鸡禽杆菌外膜蛋白p9的编码碱基序列,将其接入表达载体pNZ8149的多克隆位点并导入食品级乳酸乳球菌NZ3900。将重组乳酸乳球菌灭活后经肌肉注射免疫SPF鸡,免疫后攻毒测定乳酸乳球菌的保护效果,检测血清中IgG抗体和鼻黏膜sIgA抗体水平并检验免疫效果。PCR结果显示,成功构建了含p9片段的重组乳酸乳球菌,Western-blot检测显示,p9片段可在重组乳酸乳球菌内表达。重组菌最佳诱导表达条件：Nisin浓度为20 ng/mL、诱导时间为4 h。鸡肌肉注射免疫后可产生p9蛋白特异性的血清Ig G和鼻黏膜sIgA抗体。乳酸乳球菌重组疫苗免疫鸡对副鸡禽杆菌攻毒的保护率为60%-80%。结果表明,本研究成功构建了重组乳酸乳球菌pNZ8149-p9/NZ3900,该重组菌作为免疫原免疫鸡,对副鸡禽杆菌攻毒具有较好的免疫保护作用。

To construct a recombinant Lactobacillus laccoccus that can express the outer membrane protein of Avibacterium paragallinarum（Apg）.The p9 gene,which is part of encoding Apg outer membrane protein,was optimum synthesed according to the preference of Lactococcus lactis expression,and connected into the multiple cloning site of p NZ8149 expression vector.The recombinant vector was transformed into food-grade Lactobacillus lactobacillus NZ3900.Then,the recombinant Lactobacillus laccoccus was inactivated and injected SPF chicken by chest muscle.The test chickens were challenged by Apg to check the immune efficiency.Ig G and s Ig A were tested to check antibody level.The PCR result showed that the p9 gene was inserted into p NZ8149/NZ3900 successfully.Western-blot results showed the recombinant plasmid could be expressed in Lactobacillus lactobacillus,optimizing inducement time was4 h and the best concentration of inducer Nisin was 20 ng/mL.Serum Ig G and mucosa s Ig A could be detected after being inoculated by the vaccine made fromL.lactis pNZ8149-p9/NZ3900.The injected chickens about from 60% to 80% were protected from Apg challenges.In conclusions,the recombinant Lactobacillus laccoccus with p NZ8149-p9/NZ3900 was successfully conducted,and the chickens inoculated with it showed perfect protection against Apg challenge.