表达猪传染性胃肠炎病毒SLN基因的重组乳酸乳球菌对仔猪安全性及免疫效力研究

为了检验青岛农业大学预防兽医学重点实验室构建的能够表达猪传染性胃肠炎SLN基因的乳酸乳球菌对仔猪的安全性及免疫效力,试验以口服的方式给试验猪免疫重组菌,监测免疫接种后试验猪体重、临床症状及肠道内正常菌群的变化;给小鼠等非靶标性动物口服该重组菌,检验其安全性,同时利用间接酶联免疫吸附试验(ELISA)方法监测仔猪体内特异性IgG抗体的变化情况。结果表明:试验仔猪口服免疫重组菌后体重与肠道内正常菌群不受影响,重组菌对仔猪及非靶动物没有致病性,且不会向环境微生物转移; ELISA检测该重组菌免疫仔猪后特异性IgG抗体与免疫前相比有明显差异。说明重组SLN基因的乳酸乳球菌对于预防猪传染性胃肠炎是安全有效的。

Construction and Immunogenicity of Recombinant Lactococcus lactis Expressing S1 Protein of Porcine Epidemic Diarrhea Virus(PEDV)

To evaluate the specific immune responses induced by recombinant Lactococcus lactis（L.lactis） which expresses porcine epidemic diarrhea virus（PEDV） S1 protein through oral administration,the spike gene fragment of PEDV was amplified from PEDV SDLY strain to construct p MG36 e-S1 recombinant plasmid.The recombinant plasmid was then electro-transferred into competent cells of L.lactis MG1363,to prepare the recombinant L.lactis expressing S1 protein of PEDV.The expression of target protein was identified by SDS-PAGE and Western-blot.New Zealand white rabbits were orally administered with the recombinant strain;the antibody titer in intestinal mucosa and serum was detected by neutralizing test;and the specific Ig G in serum was evaluated by indirect ELISA.The results showed that the recombinant L.lactis could effectively induce high level of Ig G in serum and high level of mucosal immune antibody.The recombinant L.lactis is qualified to be a potential oral vaccine because it could successfully stimulate both humoral and mucosal immune responses against PEDV.

猪流行性腹泻病毒山东分离株SD-2016的S基因序列分析

为研究本实验室分离的SD-2016分离株S基因序列特点,设计扩增猪流行性腹泻病毒（PEDV）S基因的2对引物,将扩增出的目的片段与克隆载体连接后测序,利用分子生物学软件对目的基因的核苷酸序列和氨基酸序列进行同源性比对分析。结果：SD-2016毒株与CV777、LZC等GI群的毒株在核苷酸序列同源性比对结果为93.8%~95.0%,氨基酸序列同源性比对结果在92.5%~94.1%。绘制系统发育进化树发现,SD-2016株与中国广东分离株GD12、湖南分离株HUN在一个分支上,亲缘关系较近。对S基因的中和表位进行分析发现主要的氨基酸位点突变集中于抗原表位区COE区域（第499-638位氨基酸）和SS6（第764-771位氨基酸）,分别有6个位点和2个位点的突变。通过对SD-2016分离株S基因的分析,为以后猪流行性腹泻病的基因工程疫苗研制提供参考。