嗜水气单胞菌fla基因重组植物乳杆菌的构建及其表达产物的免疫原性分析

Construction of recombinant Lactobacillus plantarum with fla gene of Aeromonas hydrophila and immunogenicity analysis of its expression products

为了检测嗜水气单胞菌(Aeromonas hydrophila)鞭毛(flagellum,fla)基因在重组植物乳杆菌中表达产物的免疫原性,试验将目的基因fla与表达载体pPG结合,通过电击法将其转至植物乳杆菌感受态细胞中,构建重组植物乳杆菌Lp-pPG-fla,通过Western-blot检测重组植物乳杆菌fla蛋白的表达情况。将120尾鲫鱼随机分为Lp-pPG-fla组、Lp-pPG组和PBS组,各组分别连续口服Lp-pPGfla、Lp-pPG和PBS 28 d,分别在0(免疫前)及开始免疫后第7,14,21,28天时采集血清,通过ELISA试剂盒检测血清中免疫球蛋白(Ig)M的含量,溶菌酶(LYS)、碱性磷酸酶(AKP)、超氧化物歧化酶(SOD)的活性。在第29天(口服结束后第1天)进行免疫保护性试验,观察2周并记录死亡情况,计算免疫保护率。结果表明:成功构建了重组植物乳杆菌Lp-pPG-fla,获得的fla蛋白条带大小为38 ku。免疫后Lp-pPG-fla组鲫鱼血清中IgM含量在7,14,21,28天极显著高于其他两组(P<0.01),Lp-pPG组和PBS组之间差异不显著(P>0.05);LYS活性在14,21,28天极显著高于其他两组(P<0.01),Lp-pPG组和PBS组之间差异不显著(P>0.05);AKP活性在7,14,21,28天极显著高于其他两组(P<0.01),Lp-pPG组和PBS组之间差异不显著(P>0.05);SOD活性在14,21,28天极显著高于其他两组(P<0.01),在第7天时显著高于其他两组(P<0.05),LP-PPG组和PBS组之间差异不显著(P>0.05)。对鲫鱼攻毒后,Lp-pPG-fla组的免疫保护率达到60%,远高于对照组。说明用重组植物乳杆菌免疫鲫鱼可以提高其免疫能力。

In order to detect the immunogenicity of the expression products of flagellum(fla)gene of Aeromonas hydrophila in recombinant Lactobacillus plantarum,in the experiment,the target gene fla was combined with the expression vector pPG,and it was transferred into Lactobacillus plantarum by electric shock method to construct the recombinant Lactobacillus plantarum Lp-pPG-fla.The expression of fla protein was detected by Western-blot.120 crucian carps were randomly divided into Lp-pPG-fla group,Lp-pPG group and PBS group,and each group was continuously oral administration of Lp-pPG-fla,Lp-pPG and PBS for 28 days,respectively.Sera were collected on 0(preimmune),and 7,14,21,and 28 days after immunization;the content of IgM,and the activity of lysozyme(LYS),alkaline phosphatase(AKP),superoxidase(SOD)in sera were detected by ELISA kit.On the 29th day(the 1st day after oral administration),the challenge protection test was performed,and the mortality was recorded for 2 weeks,and the immune protection rate was calculated.The results showed that the recombinant Lp-pPG-fla was successfully constructed,and the obtained fla protein band size was 38 ku.The serum IgM content of crucian carps in LppPG-fla group was extremely significantly higher than that in the other two groups on the 7th,14th,21th and 28th day after immunization(P<0.01),and there was no significant difference between the Lp-pPG group and PBS group(P>0.05).The activity of LYS was extremely significantly higher than those in the other two groups on days 14,21,and 28(P<0.01),and there was no significant difference between the Lp-pPG group and PBS group(P>0.05).The activity of AKP was extremely significantly higher than those in the other two groups on days 7,14,21,and 28(P<0.01),and there was no significant difference between the Lp-pPG group and PBS group(P>0.05).The activity of SOD was extremely significantly higher than that in the other two groups on days 14,21,and 28(P<0.01);on the 7th day,the activity of SOD was significantly higher(P<0.05),and there was no significant difference between the Lp-pPG group and PBS group(P>0.05).After the challenge in crucian carps,the immune protection rate of Lp-pPG-fla group reached 60%,which was much higher than that of the control group.The results suggested that the immunization of the recombinant Lactobacillus plantarum to crucian carps could improve its immunity,and laid a research foundation for preventing fish infection with Aeromonas hydrophila.