CAJ | 학술논문

目的：制备与鉴定羊布鲁杆菌脂蛋白OM P19单克隆抗体，用于布菌感染免疫机制研究。方法将OM P19基因连接入pET‐30a（＋）表达载体中，构建pET‐30a（＋）／OMP19质粒，转化入大肠埃希氏菌BL21（E3），以不同浓度异丙基‐β‐D硫代半乳糖苷（IPTG）进行诱导表达，采用镍金属螯合亲和层析（NI‐NTA）纯化；以布菌阳性血清检测重组蛋白免疫反应性，并利用杂交瘤技术制备单克隆抗体，以天然OM P19蛋白及布菌外膜蛋白提取物（NM P）对制备的单克隆抗体进行Western Blot及酶免疫染色试验（IEST）鉴定。结果表达了OMP19蛋白，分子量约19 kDa ，通过纯化纯度可达95％，Western Blot分析显示蛋白具有良好的抗原性，并制备了OM P19抗原23株鼠源性单克隆抗体，鉴定结果显示22（95．65％）株能与天然OM P19蛋白反应，18（78．26％）株能与NM P反应，其中IgG1（k）亚型占91．30％；4株能与羊种布鲁氏菌菌涂片反应。结论成功制备具有良好抗原性的重组OM P19蛋白，筛选出识别天然蛋白的单克隆抗体，已初步应用于布菌的检测，为OM P19抗原B细胞表位的筛选奠定基础。
목적：제비여감정양포로간균지단백OM P19단극륭항체，용우포균감염면역궤제연구。방법장OM P19기인련접입pET‐30a（＋）표체재체중，구건pET‐30a（＋）／OMP19질립，전화입대장애희씨균BL21（E3），이불동농도이병기‐β‐D류대반유당감（IPTG）진행유도표체，채용얼금속오합친화층석（NI‐NTA）순화；이포균양성혈청검측중조단백면역반응성，병이용잡교류기술제비단극륭항체，이천연OM P19단백급포균외막단백제취물（NM P）대제비적단극륭항체진행Western Blot급매면역염색시험（IEST）감정。결과표체료OMP19단백，분자량약19 kDa ，통과순화순도가체95％，Western Blot분석현시단백구유량호적항원성，병제비료OM P19항원23주서원성단극륭항체，감정결과현시22（95．65％）주능여천연OM P19단백반응，18（78．26％）주능여NM P반응，기중IgG1（k）아형점91．30％；4주능여양충포로씨균균도편반응。결론성공제비구유량호항원성적중조OM P19단백，사선출식별천연단백적단극륭항체，이초보응용우포균적검측，위OM P19항원B세포표위적사선전정기출。
We produced and identified the monoclonal antibodies against Brucella melitensis U‐lipoprotein OMP19 .A DNA fragment coding omp19 of Brucella melitensis was amplified by PCR ,and inserted into the vector of pET‐30a(+ ) ,the result‐ant recombinant plasmid ,which we designated as pET‐30a(+ )/omp19 .We then transformed the plasmid into BL21(DE3) competent cells for the expression of the OMP19 protein .After induction with different concentrations of IPTG ,the colleted cells were analyzed by SDS‐PAGE ,and then OMP19 monoclonal antibodies were prepared through hybridoma technology . These mAbs were tested to reactivity to rOMP19 and nature membrance proteins (NMP) of Brucella melitensis by Western blot and IEST .We successfully constructed an expression vector of pET 30a(+ )/omp19 .An IPTG‐induced expression of the OMP19 protein (19 kDa in molecular weight) was demonstrated by SDS‐PAGE .The fusion protein existed in the form of solu‐ble ,and the OMP19 protein of high purity could be obtained by Ni‐NTA .Western blot assay showed that the refolded protein could be recognized by the anti‐serum against Brucella melitensis .Twenty‐three mAbs to OMP19 was produced in which 91 .30% were IgG1 ,twenty‐two (95 .65% ) mAbs could recognize nature OMP19 protein ,and eighteen (78 .26% ) mAbs could recognize NMP ,four mAbs could react with Brucella melitensis .The protein maintained good immunogenicity and twenty‐three mAbs were obtained ,which we believe provides a good protein candidate for the immunological research .