中华传染病杂志
中華傳染病雜誌
중화전염병잡지
CHINESE JOURNAL OF INFECTIOUS DISEASES
2009年
9期
517-521
,共5页
李存枚%邓松华%曹洁%王锦红%陈璐%黄德圣%潘卫
李存枚%鄧鬆華%曹潔%王錦紅%陳璐%黃德聖%潘衛
리존매%산송화%조길%왕금홍%진로%황덕골%반위
HIV-1%基因产物%tat%重组蛋白质类%半胱氨酸%基因表达%免疫%大肠埃希菌
HIV-1%基因產物%tat%重組蛋白質類%半胱氨痠%基因錶達%免疫%大腸埃希菌
HIV-1%기인산물%tat%중조단백질류%반광안산%기인표체%면역%대장애희균
HIV-1%Gene products%tat%Recombinant proteins%Cysteine%Gene expression%Immunity%Escherichia coli
目的 构建HIV-1 HXB2株Tat基因半胱氨酸富集区3'末端移位突变体,经原核表达和纯化,分析该突变体蛋白(Tat-cct)的免疫原性.方法 用PCR方法将HIV-1 HXB2株Tat基因的半胱氨酸富集区(64～111位核苷酸)移位至Tat基因的3'末端,获得其突变体DNA序列,并构建其原核表达质粒pET32a-Tat-cct,转入大肠埃希菌E.coli BL21(DE3)中进行诱导表达及纯化.以Tat-cct融合表达蛋白免疫BALB/c小鼠,ELISA方法对该抗血清进行免疫原性检测分析.结果 pET32a-Tat-cct重组质粒可在E.coli BL21(DE3)中诱导表达,纯化后其融合蛋白相对分子质量约为31 000.Tat-cct重组蛋白免疫小鼠诱导产生的抗体滴度为1:1600,该抗体与Tat-cct蛋白和Tat蛋白(1～101位氨基酸)均呈特异性结合反应.结论 Tat突变体重组蛋白Tat-cct能在大肠埃希菌中有效表达.并较好保留其免疫原性,为HIV-1 Tat疫苗的基础研究提供了有价值的实验结论.
目的 構建HIV-1 HXB2株Tat基因半胱氨痠富集區3'末耑移位突變體,經原覈錶達和純化,分析該突變體蛋白(Tat-cct)的免疫原性.方法 用PCR方法將HIV-1 HXB2株Tat基因的半胱氨痠富集區(64～111位覈苷痠)移位至Tat基因的3'末耑,穫得其突變體DNA序列,併構建其原覈錶達質粒pET32a-Tat-cct,轉入大腸埃希菌E.coli BL21(DE3)中進行誘導錶達及純化.以Tat-cct融閤錶達蛋白免疫BALB/c小鼠,ELISA方法對該抗血清進行免疫原性檢測分析.結果 pET32a-Tat-cct重組質粒可在E.coli BL21(DE3)中誘導錶達,純化後其融閤蛋白相對分子質量約為31 000.Tat-cct重組蛋白免疫小鼠誘導產生的抗體滴度為1:1600,該抗體與Tat-cct蛋白和Tat蛋白(1～101位氨基痠)均呈特異性結閤反應.結論 Tat突變體重組蛋白Tat-cct能在大腸埃希菌中有效錶達.併較好保留其免疫原性,為HIV-1 Tat疫苗的基礎研究提供瞭有價值的實驗結論.
목적 구건HIV-1 HXB2주Tat기인반광안산부집구3'말단이위돌변체,경원핵표체화순화,분석해돌변체단백(Tat-cct)적면역원성.방법 용PCR방법장HIV-1 HXB2주Tat기인적반광안산부집구(64～111위핵감산)이위지Tat기인적3'말단,획득기돌변체DNA서렬,병구건기원핵표체질립pET32a-Tat-cct,전입대장애희균E.coli BL21(DE3)중진행유도표체급순화.이Tat-cct융합표체단백면역BALB/c소서,ELISA방법대해항혈청진행면역원성검측분석.결과 pET32a-Tat-cct중조질립가재E.coli BL21(DE3)중유도표체,순화후기융합단백상대분자질량약위31 000.Tat-cct중조단백면역소서유도산생적항체적도위1:1600,해항체여Tat-cct단백화Tat단백(1～101위안기산)균정특이성결합반응.결론 Tat돌변체중조단백Tat-cct능재대장애희균중유효표체.병교호보류기면역원성,위HIV-1 Tat역묘적기출연구제공료유개치적실험결론.
Objective To construct shifting mutant of cysteine-rich region to 3?@terminal of Tat gene of human immunodeficiency virus type-1 (HIV-1) HXB2 strain, and to analyze the immunogenicity of mutant protein (Tat-cct) after prokaryotically expressed and purified. Methods The cysteine-rich region (nucleotides 64--111) of Tat gene was shifted to 3'terminal of Tat of HIV-1 HXB2 strain by polymerase chain reaction (PCR) and Tat mutant DNA sequence was obtained. Prokaryotie express plasmid pET32a-Tat-cct was constructed and transformed into E. coli BL21 (DE3), then Tat-cct protein was expressed and purified. BALB/c mice were immunized with the fusion protein Tat-cct, and immunogenicity of the immunized serum was detected by enzyme-linked immunosorbent assay (ELISA). Results The recombinant plasmid pET32a-Tat-cct expressed in E. coli BL21 (DE3) and the relative molecular mass of the purified fusion protein was 31 000. The serum antibody titer of mice immunized with Tat-cct recombinant protein was 1 : 1600, which binded specifically with both Tat-ect protein and Tat protein (amino acids 1-101). Conclusions The recombinant protein Tat-cct of Tat mutant strain can be expressed efficiently in E. coli and well retains immunogenicity, which provides valuable information for basic research of HIV-1 Tat vaccine.
