中华皮肤科杂志
中華皮膚科雜誌
중화피부과잡지
Chinese Journal of Dermatology
2012年
6期
396-399
,共4页
龙福泉%王千秋%张津萍%龚匡隆
龍福泉%王韆鞦%張津萍%龔劻隆
룡복천%왕천추%장진평%공광륭
密螺旋体,苍白%Tp0136%基因表达%膜蛋白质类%免疫活性
密螺鏇體,蒼白%Tp0136%基因錶達%膜蛋白質類%免疫活性
밀라선체,창백%Tp0136%기인표체%막단백질류%면역활성
Treponema pallidum%Tp0136%Gene expression%Membrane proteins%Immunocompetence
目的 克隆并表达梅毒螺旋体Tp0136基因,对其表达产物进行纯化以及免疫活性分析.方法 全基因合成Tp0136基因,构建E,coli表达载体；诱导表达并纯化重组蛋白,用SDS-PAGE和Western 印迹鉴定.用纯化的重组蛋白Tp0136免疫新西兰兔,并以重组Tp0136作为包被抗原建立间接酶联免疫吸附试验( ELISA)以鉴定其免疫原性,Western印迹检测梅毒阳性血清鉴定其免疫反应性.结果 成功构建E.coli表达载体pET28a-Tp0136,并表达和纯化出膜蛋白Tp0136,相对分子质量为50 000,纯度＞95％.用重组蛋白Tp0136免疫新西兰兔,能刺激其产生高滴度抗体,具有较强的免疫原性.Western印迹显示其能与梅毒阳性血清发生特异性反应,具有较好的免疫反应性.结论 成功表达具有Tp0136全基因和较强免疫活性的膜蛋白,Tp0136可能在梅毒螺旋体的免疫致病机制中起到重要作用.
目的 剋隆併錶達梅毒螺鏇體Tp0136基因,對其錶達產物進行純化以及免疫活性分析.方法 全基因閤成Tp0136基因,構建E,coli錶達載體；誘導錶達併純化重組蛋白,用SDS-PAGE和Western 印跡鑒定.用純化的重組蛋白Tp0136免疫新西蘭兔,併以重組Tp0136作為包被抗原建立間接酶聯免疫吸附試驗( ELISA)以鑒定其免疫原性,Western印跡檢測梅毒暘性血清鑒定其免疫反應性.結果 成功構建E.coli錶達載體pET28a-Tp0136,併錶達和純化齣膜蛋白Tp0136,相對分子質量為50 000,純度＞95％.用重組蛋白Tp0136免疫新西蘭兔,能刺激其產生高滴度抗體,具有較彊的免疫原性.Western印跡顯示其能與梅毒暘性血清髮生特異性反應,具有較好的免疫反應性.結論 成功錶達具有Tp0136全基因和較彊免疫活性的膜蛋白,Tp0136可能在梅毒螺鏇體的免疫緻病機製中起到重要作用.
목적 극륭병표체매독라선체Tp0136기인,대기표체산물진행순화이급면역활성분석.방법 전기인합성Tp0136기인,구건E,coli표체재체；유도표체병순화중조단백,용SDS-PAGE화Western 인적감정.용순화적중조단백Tp0136면역신서란토,병이중조Tp0136작위포피항원건립간접매련면역흡부시험( ELISA)이감정기면역원성,Western인적검측매독양성혈청감정기면역반응성.결과 성공구건E.coli표체재체pET28a-Tp0136,병표체화순화출막단백Tp0136,상대분자질량위50 000,순도＞95％.용중조단백Tp0136면역신서란토,능자격기산생고적도항체,구유교강적면역원성.Western인적현시기능여매독양성혈청발생특이성반응,구유교호적면역반응성.결론 성공표체구유Tp0136전기인화교강면역활성적막단백,Tp0136가능재매독라선체적면역치병궤제중기도중요작용.
Objective To clone and express the Tp0136 gene of Treponema pallidum,to purify the recombinant protein and to evaluate its immunocompetence.Methods The full-length Tp0136 gene was synthesized,then subcloned into the expression vector pET28a to construct a recombinant plasmid,pET28a-Tp0136,which was subsequently transfected into E.coli Rosetta for protein expression.The recombinant protein was purified with nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography,and identified by using sodium dodecyl sulfate polyacrylamide gel electropheresis (SDS-PAGE) and Western blot.New Zealand rabbits were immunized with the recombinant Tp0136 (rTp0136)protein,and anti-Tp0136 polyclonal antibodies in sera of the rabbits were examined by indirect enzyme linked immunosorbent assay (ELISA) with rTp0136 protein as the coating antigen.Also,positive sera were obtained from patients with syphilis and examined by Western blot to identify the immunoreactivity of the rTp0136 protein.Results The E.coli expression vector pET28a-Tp0136 was constructed successfully,and rTp0136 protein was also successfully expressed with a molecular weight of about 50 kD and a purity above 95％.High titres of anti-rTp0136 antibodies were detected in sera of rabbits immunized with the rTp0136 protein,and Western blot showed that the rTp0136 could specifically react with the sera from syphilitic patients,which proved the high immunogenicity and immunoreactivity of the recombinant protein.Conclusions The full-length Tp0136 membrane protein is successfully expressed with a high immunocompetence,and Tp0136 membrane protein may play an important role in the pathogenic mechanisms of Treponema pallidum.
