ETEC 987P、K88二价重组干酪乳酸菌的构建及免疫仔猪效果评价  

Construction of ETEC 987P and K88 bivalent recombinant Lacticaseibacillus casei and evaluation of its immune effect in piglets

作　　者：杨雨晴 李颖 蔡昌福 乔志刚 王桂华[1] 王北艳[1] 余丽芸[1] 侯喜林[2] YANG Yuqing;LI Ying;CAI Changfu;QIAO Zhigang;WANG Guihua;WANG Beiyan;YU Liyun;HOU Xilin(College of Life Science and Biotechnology,Heilongjiang Bayi Agricultural University,Daqing 163319,China;College of Animal Science and Veterinary Medicine,Heilongjiang Bayi Agricultural University,Daqing 163319,China)

机构地区：[1]黑龙江八一农垦大学生命科学技术学院,黑龙江大庆163319 [2]黑龙江八一农垦大学动物科技学院,黑龙江大庆163319

出　　处：《黑龙江畜牧兽医》2023年第14期102-107,139,共7页Heilongjiang Animal Science And veterinary Medicine

基　　金：黑龙江省高校科技成果产业化前期研发培育项目(1253CGZH14);大庆指导性科技计划项目(zd-2020-62);黑龙江八一农垦大学研究生创新项目(YJSCX2019-Y29);黑龙江省创新训练项目(202110223055)。

摘　　要：为了制备产肠毒素大肠杆菌(enterotoxigenic Escherichia coli,ETEC)987P和K88的二价重组乳杆菌,并检测其免疫效果,试验根据ETEC C83916株(GenBank登录号M35257.1)987P基因序列和ETEC C83902株(GenBank登录号M29375.1)K88基因序列设计合成特异性引物,将PCR扩增的987P、K88基因连接至穿梭载体pHCE1LB-pgsA(简称pLA),热激法转化至大肠杆菌BL21感受态细胞中,经PCR、双酶切鉴定为阳性的重组质粒电转化入干酪乳杆菌中,通过Western-blot、间接免疫荧光检测目的蛋白的表达情况。将50头健康的初生仔猪分为试验组和对照组,试验组初生仔猪通过口服免疫重组干酪乳杆菌菌液(浓度为1×10^(9) cfu/mL),对照组口服等体积生理盐水,连用5 d,在免疫前称量体重,并于免疫前(第0天)及免疫后第5,10,15,20,25天采集血液和粪便,采用ELISA检测血清IgG抗体和粪便中sIgA抗体水平;免疫后10天试验组和对照组分别随机选取20头仔猪用ETEC C83916株和C83902株进行攻毒,计算攻毒保护率,试验结束后称量仔猪体重。结果表明:成功构建了重组菌pLA-987P-K88/L.casei,Western-blot检测显示pLA-987P-K88/L.casei在85 ku处出现明显条带,在荧光显微镜下pLA-987P-K88/L.casei可观察到明显的绿色荧光。初生仔猪免疫重组菌pLA-987P-K88/L.casei后5~25 d,IgG抗体和sIgA抗体水平显著或极显著升高(P<0.05或P<0.01或P<0.001)。重组菌对ETEC C83916的攻毒保护率为90%,对ETEC C83902的攻毒保护率为80%。说明ETEC 987P、K88二价重组干酪乳酸菌可以有效抑制仔猪感染ETEC,大大降低新生仔猪腹泻率。In order to prepare the divalent recombinant Lacticaseibacillus producing 987P and K88 of enterotoxigenic Escherichia coli(ETEC),and to detect its immune effect,in this experiment,specific primers were designed and synthesized according to the gene sequences of 987P of ETEC C83916(GenBank M35257.1)and K88 of ETEC C83902(GenBank M29375.1).987P and K88 genes amplified by PCR were connected into the shuttle vector pHCE1LB-pgsA(pLA for short),which was transformed into Escherichia coli BL21 competent cells by heat shock method.Recombinant plasmids identified as positive by PCR and double enzyme digestion identification were electro-transformed into Lacticaseibacillus casei,and the expression of the interest protein was detected by Westerm-blot and indirect immunofluorescence.50 healthy newborn piglets were divided into experimental and control groups;the newborn piglets in the experimental group were orally immunized with recombinant Lacticaseibacillus casei solution(concentration of 1×10^(9) cfu/mL),and the control group was orally administered the same amount of normal saline,for consecutive 5 days.They were weighed before immunization,and blood and feces were collected before immunization(day 0)and days 5,10,15,20,and 25 after immunization.ELISA was used to detect the levels of serum IgG antibody and slgA antibody in feces.Each 20 piglets were randomly selected in the experimental group and the control group for challenge with ETEC C83916 and C83902 strains 10 days after immunization,and the protection rate after challenge was calculated.The piglets were weighed after the experiment.The results showed that the recombinant bacteria pLA-987P-K88/L.casei was successfully constructed.Western-blot detection showed that pLA-987PK88/L.casei showed a distinct band at 85 ku.Under fluorescence microscopy,pLA-987P-K88/L.casei could observe a distinct green fluorescence.5-25 days after the immunization recombinant bacteria pLA-987P-K88/L.casei in newborn piglets,the levels of IgG antibody and slgA antibody increased signific

关 键 词：重组干酪乳杆菌 产肠毒素大肠杆菌 987P K88 仔猪 免疫效果 

分 类 号：S852.4[农业科学—基础兽医学]