1、方法 提取无乳链球菌临床分离菌株基因组DNA,根据GenBank公布的无乳链 球菌sip基因序列，应用DNAStar软件Protean程序预测sip基因抗原表位、亲水性和 抗原性指数等参数，并设计引物，PCR扩增出sip抗原表位基因.将其插入至表达质 粒pET-30（a）的多克隆位点（MCS）,构建重组表达质粒sippET,以常规转化方法将 sip-pET转入E.coli/BL21（DE3）, IPTG诱导表达并优化诱导表达条件，应用SDS-PAGE 电泳分析表达蛋白图谱，Western-blot技术验证重组蛋白的免疫活性。运用Ni”亲和层 析法在自然条件下纯化重组蛋白，并建立间接ELISA方
2、法，应用该方法对无乳链球 菌全菌体裂解免疫制剂免疫的家兔血清抗体滴度进行检测。结果 结果经测序和酶切分析表明，克隆获得了无乳链球菌sip抗原表位编码序 列，且插入片段读码框架正确，原核表达重组质粒构建成功。经IPTG诱导.BL2KDE3） /sip-pET系统表达出相对分子质量为40 KD的可溶性融合蛋白（表达量占菌体总量的 43.8%）. Western-blot证实了重组蛋白的免疫活性。纯化后蛋白含量为5.09mg/ml,间 接ELISA方法检测3次免疫后58 d的血清免疫组抗体滴度达到1:3200,免疫70 d后 仍保持较高抗体滴度.结论 成功克隆牛源无乳链球菌sip抗原表位序列，该序列
3、897 bp,编码299个 氨基酸残基，构建的重组表达载体在大肠杆菌中获得高效可溶性表达，并建立了检测 无乳链球菌免疫制剂免疫抗体滴度的间接ELISA方法，为进一步研究SIP抗原表位 重组蛋白免疫原性及无乳链球菌疫苗免疫效果研究奠定了基础。关键词：牛源无乳链球菌；sip基因；原核表达；纯化：间接ELISA；抗体检测Study on the Prokaryotic expression and PreliminaryApplication of Coding Region Gene of Sip Epitope ofStreptococcus agalactiae From BovineAbst
4、ractObjectiv lb construct prokaryotic expression vector of sip gene,express it in Escherichia coli BL21,and study the immunocompetence and application.Methods Genomic DNA was extracted from streptococcus agalactiae,epitope sites, hydrophilicity and antigenic index parameters were predicted by DNASta
5、r Protean program according to the relevant nucleotide sequence from GenBankand a pair of specific primers was designed to amplify the epitope of sip gene by PCR.The fragment was cloned into the multiple cloning site （MCS） of pET30（a）,fbnning the recombinant prokaryotic expression vector （sip-pET）.T
6、he recombinant plasmid sip-pET was transformed into E.coli BL21（DE3） by common method and the BL21（DE3）/pET was induced by Isopropyl-p-D-thioglactoside （IPIG） to express fusion protein,optimize the expression condition.Then the expressed proteins in E.coli BL21（DE3） were analyzed with SDS-PAGE elect
7、rophoresis.Western blotting was performed to identify the Immune active fusion protein.The recombinant protein was purified with immobilized nicke ion affinity chromatography,and an indirect enzymelinked immunosorbent assay was developed.Taking this method to test the serum titers of vaccinated rabb
8、its which were immunized with immune pharmaceutics of streptococcus agalactiae.Results The recombinant plasmid （sip-pET） was obtained and its opening read framORF） was verified by sequence analysis.The results of SDS-PAGE showed that there was a noval protein band,which was 40KD.Westem blotting assa
9、y proved that the fusion protein could be specifically recognized by anti-Str.agalactiae antibody.The density of Sip protein was 5.09mg/ml after purification,the antibody valence of streptococcus agalactiae were 1:3200 when tested by indirect enzymelinked immunosorbent assay,Specific antibody could
10、be detected in 70 days after injection immunization.Conclusions These results showed that coding region gene of sip epitope of streptococcus agalactiae from bovine was successfully cloned, the sequence was composed of 897 nucleotides encoding a polypetide of 299 amino acids,prokaryotic expression ve
11、ctor was efficiently expressed in E.coli BL21（DE3） and an indirect enzymelinked immunosorbent assay for detection of Bovine Mastitis Streptococcus agalactiae was developed.The previous work has not only roll out a basis for the studies of antigenicity identification of the recombinant protein,but al
12、so laid a necessary foundation for future research on possible Immune effect of streptococcus agalactiae in the future.Key words: Bovine source Streptococcus agalactiae;sip gene;Prokaryotic expression: Purification;Indirect enzymelinked immunosorbent assayDirected by: prof. Bu Rie （Ph. D）AppI icant
13、for Master degree： LANG Ji ng min （preventive veterinary Science）（College of Animal Science and Technology,!nner Mongolia University fbr Nationalities,Tongliao 028043, China）英文缩写词表英文缩写英文名称中文名称4CN4-Chloro 1-naphthol四氯奈酚A492Absorption value at 492nm492纳米处的吸光值AmpAmpicillin氨千青霉素APSAmmonium persulfate过硫酸
14、铉bpBase pair碱基对CPCaspsular polysaccharide荚膜多糖CSFColony-stimulating factor集落刺激因子DNAdeoxyribose nucleic acid脱氧核糖核酸dNTPDeoxynucleoside triphosphate脱氧核吾三磷酸DTTDithiothreitol_硫苏糖醇E.coliEscherichia coli大肠杆菌E.coli BL2IEscherichia coli BL21基因程表达菌E.coliJM109Escherichia coli JM109基因1程克隆菌EBEthidium Bromide漠化乙锭E
15、D1Aethylene diamine tetraacetic acid乙一胺四乙酸ELISAEnzyme-linked immunosorbent assay酶联免疫吸附实验EPEppendorf离心管FCAFreund complete adjuvant福氏完全佐剂FIAFreund incomplete adjuvant福氏不完全佐剂Genebank核吾酸序列数据库hHour小时HRPHorse radish peroxidase辣根过氧化物酶IFInterferon干扰素IgImmuno globulin免疫球蛋白ILInterleukin白细胞介素IPTGisopropylthio-p-D-thiogalactoside异丙基硫代半乳糖昔Kankanamycin卡那霉素KDkilodalton千道尔顿LLiter升LBLuria-Benrtani mediumLB培养基
