中华传染病杂志
中華傳染病雜誌
중화전염병잡지
CHINESE JOURNAL OF INFECTIOUS DISEASES
2012年
6期
324-329
,共6页
彭国平%赵冬久%周承%周林福%陈智
彭國平%趙鼕久%週承%週林福%陳智
팽국평%조동구%주승%주림복%진지
基因,病毒%肝炎表面抗原,乙型%细胞毒性T淋巴细胞相关抗原%基因表达%质粒%免疫活性
基因,病毒%肝炎錶麵抗原,乙型%細胞毒性T淋巴細胞相關抗原%基因錶達%質粒%免疫活性
기인,병독%간염표면항원,을형%세포독성T림파세포상관항원%기인표체%질립%면역활성
Genes,viral%Hepatitis B surface antigens%CTLA-4 antigen%Gene expression%Plasmids%Immunocompetence
目的 构建HBsAg基因(HBV S)与抗鼠细胞毒性T淋巴细胞相关抗原-4(CTLA-4)单链抗体(ScFv)的真核质粒载体,并检测融合表达蛋白特异性抗原的结合活性.方法 将CTLA-4单链抗体的真核表达质粒pSect2/ScFv4F10及含HBV S基因的质粒pSect2/S分别经SfiI和HindⅢ双酶切后,S基因片段进一步克隆至质粒pSect2/ScFv4F10中.将质粒pSect2/ScFv40、pSect2/S-ScFv4F10转染中华仓鼠卵巢细胞,Western印迹法检测目的蛋白的表达.将表达的蛋白经超滤浓缩与亲和层析纯化后,分别通过竞争抑制ELISA和表面等离子共振(SPR)技术,检测其与重组小鼠CTLA-4抗原的结合活性及亲合常数.采用One-way ANOVA比较组间差异.结果 成功构建可稳定表达S-ScFv4F10蛋白的真核质粒载体pSect2/S-ScFv4F10.十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)和Western印迹法检测表明,其表达蛋白的相对分子质量约为52×103.当将抗鼠CTLA 4的亲本单链抗体4F10浓度固定时,其与CTLA-4纯化抗原混合反应的吸光度(A)570值随ScFv融合蛋白比例的减少而逐步增大;且当ScFv、S-ScFv4F10与4F10单链抗体以摩尔比为2∶1混合时,其对4F10结合抗原的竞争抑制率分别可达到72.6％和64.5％;亲本单链抗体、ScFv4F10、S-ScFv4n0与重组CTLA-4抗原的结合平衡常数KA值分别为7.29×108mol/L、9.52×106mol/L和2.04×106mol/L,解离平衡常数KD分别为1.40×10-9mol/L、1.05×10-7mol/L和4.91×10-7mol/L.结论 成功构建了HBV S基因与CTLA-4单链抗体ScFv4F10的真核表达质粒载体pSect2/S-ScFv4F10,其表达的蛋白与CTLA-4抗原具有一定的亲和活性.
目的 構建HBsAg基因(HBV S)與抗鼠細胞毒性T淋巴細胞相關抗原-4(CTLA-4)單鏈抗體(ScFv)的真覈質粒載體,併檢測融閤錶達蛋白特異性抗原的結閤活性.方法 將CTLA-4單鏈抗體的真覈錶達質粒pSect2/ScFv4F10及含HBV S基因的質粒pSect2/S分彆經SfiI和HindⅢ雙酶切後,S基因片段進一步剋隆至質粒pSect2/ScFv4F10中.將質粒pSect2/ScFv40、pSect2/S-ScFv4F10轉染中華倉鼠卵巢細胞,Western印跡法檢測目的蛋白的錶達.將錶達的蛋白經超濾濃縮與親和層析純化後,分彆通過競爭抑製ELISA和錶麵等離子共振(SPR)技術,檢測其與重組小鼠CTLA-4抗原的結閤活性及親閤常數.採用One-way ANOVA比較組間差異.結果 成功構建可穩定錶達S-ScFv4F10蛋白的真覈質粒載體pSect2/S-ScFv4F10.十二烷基磺痠鈉-聚丙烯酰胺凝膠電泳(SDS-PAGE)和Western印跡法檢測錶明,其錶達蛋白的相對分子質量約為52×103.噹將抗鼠CTLA 4的親本單鏈抗體4F10濃度固定時,其與CTLA-4純化抗原混閤反應的吸光度(A)570值隨ScFv融閤蛋白比例的減少而逐步增大;且噹ScFv、S-ScFv4F10與4F10單鏈抗體以摩爾比為2∶1混閤時,其對4F10結閤抗原的競爭抑製率分彆可達到72.6％和64.5％;親本單鏈抗體、ScFv4F10、S-ScFv4n0與重組CTLA-4抗原的結閤平衡常數KA值分彆為7.29×108mol/L、9.52×106mol/L和2.04×106mol/L,解離平衡常數KD分彆為1.40×10-9mol/L、1.05×10-7mol/L和4.91×10-7mol/L.結論 成功構建瞭HBV S基因與CTLA-4單鏈抗體ScFv4F10的真覈錶達質粒載體pSect2/S-ScFv4F10,其錶達的蛋白與CTLA-4抗原具有一定的親和活性.
목적 구건HBsAg기인(HBV S)여항서세포독성T림파세포상관항원-4(CTLA-4)단련항체(ScFv)적진핵질립재체,병검측융합표체단백특이성항원적결합활성.방법 장CTLA-4단련항체적진핵표체질립pSect2/ScFv4F10급함HBV S기인적질립pSect2/S분별경SfiI화HindⅢ쌍매절후,S기인편단진일보극륭지질립pSect2/ScFv4F10중.장질립pSect2/ScFv40、pSect2/S-ScFv4F10전염중화창서란소세포,Western인적법검측목적단백적표체.장표체적단백경초려농축여친화층석순화후,분별통과경쟁억제ELISA화표면등리자공진(SPR)기술,검측기여중조소서CTLA-4항원적결합활성급친합상수.채용One-way ANOVA비교조간차이.결과 성공구건가은정표체S-ScFv4F10단백적진핵질립재체pSect2/S-ScFv4F10.십이완기광산납-취병희선알응효전영(SDS-PAGE)화Western인적법검측표명,기표체단백적상대분자질량약위52×103.당장항서CTLA 4적친본단련항체4F10농도고정시,기여CTLA-4순화항원혼합반응적흡광도(A)570치수ScFv융합단백비례적감소이축보증대;차당ScFv、S-ScFv4F10여4F10단련항체이마이비위2∶1혼합시,기대4F10결합항원적경쟁억제솔분별가체도72.6％화64.5％;친본단련항체、ScFv4F10、S-ScFv4n0여중조CTLA-4항원적결합평형상수KA치분별위7.29×108mol/L、9.52×106mol/L화2.04×106mol/L,해리평형상수KD분별위1.40×10-9mol/L、1.05×10-7mol/L화4.91×10-7mol/L.결론 성공구건료HBV S기인여CTLA-4단련항체ScFv4F10적진핵표체질립재체pSect2/S-ScFv4F10,기표체적단백여CTLA-4항원구유일정적친화활성.
Objective To construct the eukaryotic vector that expressing hepatitis B virus (HBV) S and the single fragment of variety chain (ScFv) of monoclonal antiboy against cytotoxic Tlymphocyte-associated antigen-4 (CTLA-4) and to analyze the immunological activity of recombinant S-ScFv protein.Methods The oringially constructed pSect2/ScFv4F10 and pSect2/S were double enzyme digested by Sfi I and Hind Ⅲ,respectively.Then the HBV S gene was cloned into the pSect2/ScFv4F10 vector.The pSect2/ScFv4F10 and pSect2/S-ScFv4F10 were expressed in Chinese hamster ovary (CHO) cells,and the expressed proteins were verified through sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting.After ultrafiltration concentration and affinity chromatography,the biological affinity of the expressed ScFv4F10 and S-ScFv4F10 proteins were examined by competitive enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR) technology.The comparison between groups was done by One-way ANOVA.Results The eukaryotic expression vector of pSect2/S-ScFv4F10 was successfully constructed,and relative molecular mass of the expressed protein of S-ScFv4 F10 was about 52 000 that analyzed by SDS-PAGE and Western blotting.With the fixed concentration of 4F10-mAb against CTLA-4,the A570 value of the mixed reaction with purified CTLA-4 antigen gradually increased with the decrease of ScFc fusion protein proportion; when the molar ratio of ScFv,S-ScFv4F10∶4F10=2∶1,the competitive inhibition rates against 4F10 conjugated antigen were 72.6％ and 64.5％,respectively.The affinity constants of association kinetics for CTLA-4 mAb,ScFv4F10 and S-ScFv4F10 with CTLA-4 antigen were 7.29 × 108 mol/L,9.52 × 106 mol/L and 2.04 × 106 mol/L,respectively,and the dissociation constants of KD were 1.40 × 10-9 mol/L,1.05 × 10-7 mol/L and 4.91 × 10-7 mol/L,respectively.Conclusions The eukaryotic expression vector of pSect2/S-ScFv4F10 is successfully constructed,and the recombinant protein of S-ScFv4 F10 has a fairly high affinity with CTLA-4 antigen.
