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试验旨在构建能表达牛病毒性腹泻病毒（Bovine viral diarrhea virus，BVDV）E2抗原蛋白的重组乳酸乳球菌（Lactococcus lactis），为进一步研制BVDV乳酸菌口服活载体疫苗奠定基础。将BVDV E2基因克隆后测序，根据乳酸乳球菌的密码子偏嗜性进行优化，再将优化的基因片段插入表达载体pNZ8148中，并电转化乳酸乳球菌NZ9000感受态细胞，构建重组乳酸菌pNZ8148-E2/NZ9000，经1 ng/mL乳链菌肽诱导表达后，对菌体物进行了SDS-PAGE和Western blotting分析。将重组乳酸菌pNZ8148-E2/NZ9000口服免疫6~12月龄健康犊牛，在免疫后不同时间点采集血液样品并分离血清，用间接ELISA方法检测抗体水平。结果显示，PCR扩增到了1 149 bp的目的片段，乳酸菌密码子偏嗜性优化后，GC含量从45.28%变为34.30%。重组质粒pNZ8148-E2经酶切鉴定插入片段与预期大小相符，在菌体裂解物中出现大小约42 ku的条带，与预期蛋白大小一致，且该蛋白可与BVDV E2抗体反应。在免疫犊牛的血清中检测到特异性抗BVDV E2蛋白的抗体。本研究结果表明，表达BVDV E2蛋白的重组乳酸菌口服免疫可诱导犊牛产生特异性的体液免疫反应，该重组菌具有较好的免疫原性。 相似文献
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We cloned and expressed bile salt hydrolase gene of Lactobacillus plantarum M1-UVS29 in Lactococcus lactis NZ9000 successfully. Gene-specific primers for amplification of L. plantarum bsh were designed by using sequence which availabled from Gen Bank. The production of PCR amplicon was confirmed by sequencing and cloned into pMD18-T vector, and then recombined into expression vector p NZ8148 and yielding vector p NZ8148-BSH. p NZ8148-BSH was transferred into Lactococcus lactis NZ9000. Sequencing indicated that the cloned bsh fragment contained 995 nucleotides, and shared 99.3% sequence homology with bsh gene from L. plantarum MBUL10. Cloned bsh fragment was successfully transduced into NICE expression system and confirmed by PCR and restriction digest. Recombinant BSH protein was analyzed by SDS-PAGE. The molecular weight of BSH protein was approximately 37 ku. Activity of the expressed protein was 0.77 μmol · min-1. The successfully expressed proteins by genetic engineering technology made the function of lactic acid bacteria be abundant and laid the foundation for further researches into cholesterol-lowering lactic acid bacterium food and probiotics. 相似文献
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嗜水气单胞菌外膜蛋白(OMP)在乳酸乳球菌中的表达及其对BALB/c小鼠的免疫保护效果 总被引：1，自引：0，他引：1
乳酸球菌(Lactococcus lactis)是一种安全级微生物,为活载体疫苗传递抗原的理想选择.嗜水气单胞菌(A eromonas hydrophila,Ah)能引起鱼类等多种动物的败血病,因其血清型众多,寻找共同保护性抗原是进行疫苗研究的重点.为探究嗜水气单胞菌AS1.927株外膜蛋白(OMP)在乳酸乳球菌中的表达和检测其表达产物对小鼠的免疫保护效果,本研究将己克隆的ompA基因经BamH Ⅰ和Xba Ⅰ双酶切后连接载体pNZ8048,转化乳酸乳球菌NZ9000,nisin诱导表达,并进行SDS-PAGE分析鉴定.结果显示,重组基因工程菌L.lactis[pNZ-ompA]表达的融合蛋白分子量约为36.2 kD,成熟蛋白分子量约为33.7 kD.将重组基因工程菌L.lac tis [pNZ-ompA]口服免疫BALB/c小鼠(Mus musculus),用放射免疫法检测末次免疫一周后的各组小鼠肠粘膜分泌型免疫球蛋白A(sIgA)的水平以及用间接ELISA法检测小鼠血清特异性抗体IgG,结果发现,该重组基因工程菌能显著提高sIgA的分泌(P＜0.05);同时在小鼠血清中的特异性抗体含量也显著高于对照组(P＜0.05).末次免疫两周后用100 LD50的Ah AS 1.927(3.3×105 cfu/mL)腹腔注射攻击小鼠,口服重组菌对小鼠的相对免疫保护力(relative percent survival,RPS)为87.5％,表明重组基因工程菌可诱导小鼠对AhAS1.927产生一定的免疫保护作用.该结果将为开发安全有效的口服基因工程鱼用疫苗提供一定的实验基础. 相似文献
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克隆灵芝Iz-8基因并进行原核表达,通过动物试验检测其生理活性.根据GenBank公布的Iz-8基因的DNA序列设计引物,从灵芝菌丝中提取灵芝总DNA,PCR扩增k-8基因并连接到乳酸菌食品级表达载体pNZ8149,转化乳酸乳球菌NZ3900,于30℃Nisin诱导3h,SDS-PAGE进行蛋白表达分析.将工程菌菌悬液给小鼠灌胃,对其碳廓清试验等免疫指标进行检测.结果表明:扩增到的Iz-8基因序列全长330 bp,构建了原核表达载体pNZ8149-Iz-8,LZ-8蛋白在NZ3900中得到表达.NZ3900/pNZ8149-Iz-8菌悬液对小鼠各项免疫指标有明显的调节作用. 相似文献
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通过常规鉴定和16S rDNA序列分析,从黑鲷(Sparus macrocephalus)肠道中分离鉴定出1株明串珠菌属(Leuconostoc)乳酸菌菌株;该分离株对酸、胆盐、蛋白酶具有良好耐受性,但对温度耐受能力较差;分离株培养上清液对哈维氏弧菌(Vibrio harveyi)、溶藻弧菌(Vibrio algnolyticus)、副溶血弧菌(Vibrio parahaemolyticus)的抑菌圈直径为1.12～1.22 cm,不同培养时间的抑菌效果差异无统计学意义(P＞0.05);分离株在培养24、32、40、48和56 h时的胞外多糖质量浓度为167.24～184.45 mg·L-1,不同取样时间的胞外多糖质量浓度差异无统计学意义(P＞0.05);灌服胞外多糖20 d后黑鲷血液的红细胞、白细胞数量和血红素浓度、淋巴细胞比率均显著升高(P＜0.05).综上,该分离株为产胞外多糖的明串珠属乳酸菌,可作为开发海水鱼用微生态制剂的候选菌株. 相似文献
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将猪流行性腹泻病毒LJB/03株S基因上编码中ps420(1 495～1 916 bp)基因片段插入乳酸乳球菌pNZ8112中,构建了重组表达载体pNZ8112-ps420,将其电转化入乳酸乳球菌Lactococcus lactis NZ9000,获得了表达猪流行性腹泻病毒S基因ps420的重组乳酸乳球菌,经乳链菌肽(... 相似文献
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ZHOU Yu-zhao MA Zhi-liang SUN Ting-ting YANG Jie ZHANG Xiao-miao CHAI Jun ZHANG Na-na ZHANG Yi-fang 《中国畜牧兽医》2015,42(11):2880-2887
The present study was designed to construct recombinant plasmids,which could express porcine reproductive and respiratory syndrome virus (PRRSV) ORF5 gene.RNA was extracted from spleen and lung samples of the suspected pigs which were infected with PRRSV.According to PRRSV ORF5 gene,a pair of primers was designed for RT-PCR amplification.The ORF5 target gene was cloned into pMD19-T vector and then the recombinant pMD19-ORF5 was achieved.According to the sequencing results and the characteristics of expression vectors,a pair of primers with NcoⅠand XbaⅠenzyme cleavage sites was designed.Target fragment dORF5 was amplified and then connected to pProEXHTb and pNZ8149 vectors,respectively.And recombinant HTb-dORF5/DE3 and pNZ8149-dORF5/NZ3900 was induced by IPTG and Nisin,respectively,and analyzed by SDS-PAGE and Western blotting.Recombinant HTb-dORF5/DE3 induced by 1.5 mmol/L IPTG was expressed in the highest quantity.There were specific band at about 22 ku with reactionogenicity when it was tested by SDS-PAGE and Western blotting.Recombinant pNZ8149-dORF5/NZ3900 induced by 20 ng/mL Nisin was expressed in the highest quantity.There were specific band at about 19 ku with reactionogenicity when it was tested by SDS-PAGE and Western blotting.The IFA result showed specific green fluorescence.This study successfully constructed recombinant plasmids HTb-dORF5 and pNZ8149-dORF5 and expressed,the result laid a solid foundation for further development of PRRS vaccines. 相似文献
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通过牛津杯法,从250株分离自开菲尔颗粒的乳酸菌中筛选出1株有较好抑菌活性的菌株,通过对个体特征、生理生化试验、16SrDNA序列对比,确定此株菌为乳酸乳球菌,命名为L11。在排除有机酸和过氧化氢的干扰后,发酵上清液对金黄色葡萄球菌还有抑菌活性,该菌株经过胃蛋白酶、胰蛋白酶处理后,发酵液的抑菌活性没有明显变化,说明抑菌物质为非蛋白成分,属于类细菌素,它具有较好的热稳定性,对蛋白酶不敏感,且具有较广的抗菌谱。 相似文献
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