南方医科大学学报
南方醫科大學學報
남방의과대학학보
JOURNAL OF SOUTHERN MEDICAL UNIVERSITY
2015年
2期
174-178
,共5页
赵蓓蓓%姜玉新%刁吉东%李娜%陆维%李朝品
趙蓓蓓%薑玉新%刁吉東%李娜%陸維%李朝品
조배배%강옥신%조길동%리나%륙유%리조품
屋尘螨%融合肽疫苗%1类变应原%原核表达%T细胞表位%IgE结合试验
屋塵螨%融閤肽疫苗%1類變應原%原覈錶達%T細胞錶位%IgE結閤試驗
옥진만%융합태역묘%1류변응원%원핵표체%T세포표위%IgE결합시험
Dermatophagoides pteronyssinus%fused peptide vaccine%major group 1 allergen%prokaryotic expression%T cell epitope%IgE-binding assay
目的:构建经MHCⅡ通路的屋尘螨1类变应原Der p 1的T细胞表位肽疫苗重组载体。方法分别合成TAT、IhC和含编码Der p 1的3段T细胞表位的融合核苷酸序列,用特异性引物PCR扩增相应的基因片段,分别用相应的双酶切后,用T4 DNA连接酶连接形成TAT-IhC-Der p 1-3T融合基因,并插入至原核表达载体pET-28a(+)中,构建重组原核表达载体pET-28a(+)-TAT-IhC-Der p 1-3T,Bam HⅠ和XhoⅠ进行双酶切和测序鉴定。重组载体转化大肠杆菌E. coli BL21(DE3)菌株,IPTG诱导后,经SDS-PAGE电泳分析和Western blot验证,纯化TAT-IhC-Der p 1-3T蛋白后进行IgE结合试验。结果双酶切和测序结果表明,成功构建了pET-28a-TAT-IhC-Der p 1-3T重组原核表达载体;SDS-PAGE电泳分析显示TAT-IhC-Der p 1-3T可诱导表达;Western blot检测表明该融合蛋白纯化成功;IgE结合试验表明TAT-IhC-Der p 1-3T结合屋尘螨过敏病人血清IgE的能力强于Der p 1变应原(P<0.001)。结论成功构建了可表达经MHC通路的编码Der p 1的3段T细胞表位的重组pET-28a-TAT-IhC-Der p 1-3T载体,纯化的TAT-IhC-Der p 1-3T具有较强的IgE结合能力,从而为后续经MHC通路的特异性免疫治疗奠定基础。
目的:構建經MHCⅡ通路的屋塵螨1類變應原Der p 1的T細胞錶位肽疫苗重組載體。方法分彆閤成TAT、IhC和含編碼Der p 1的3段T細胞錶位的融閤覈苷痠序列,用特異性引物PCR擴增相應的基因片段,分彆用相應的雙酶切後,用T4 DNA連接酶連接形成TAT-IhC-Der p 1-3T融閤基因,併插入至原覈錶達載體pET-28a(+)中,構建重組原覈錶達載體pET-28a(+)-TAT-IhC-Der p 1-3T,Bam HⅠ和XhoⅠ進行雙酶切和測序鑒定。重組載體轉化大腸桿菌E. coli BL21(DE3)菌株,IPTG誘導後,經SDS-PAGE電泳分析和Western blot驗證,純化TAT-IhC-Der p 1-3T蛋白後進行IgE結閤試驗。結果雙酶切和測序結果錶明,成功構建瞭pET-28a-TAT-IhC-Der p 1-3T重組原覈錶達載體;SDS-PAGE電泳分析顯示TAT-IhC-Der p 1-3T可誘導錶達;Western blot檢測錶明該融閤蛋白純化成功;IgE結閤試驗錶明TAT-IhC-Der p 1-3T結閤屋塵螨過敏病人血清IgE的能力彊于Der p 1變應原(P<0.001)。結論成功構建瞭可錶達經MHC通路的編碼Der p 1的3段T細胞錶位的重組pET-28a-TAT-IhC-Der p 1-3T載體,純化的TAT-IhC-Der p 1-3T具有較彊的IgE結閤能力,從而為後續經MHC通路的特異性免疫治療奠定基礎。
목적:구건경MHCⅡ통로적옥진만1류변응원Der p 1적T세포표위태역묘중조재체。방법분별합성TAT、IhC화함편마Der p 1적3단T세포표위적융합핵감산서렬,용특이성인물PCR확증상응적기인편단,분별용상응적쌍매절후,용T4 DNA련접매련접형성TAT-IhC-Der p 1-3T융합기인,병삽입지원핵표체재체pET-28a(+)중,구건중조원핵표체재체pET-28a(+)-TAT-IhC-Der p 1-3T,Bam HⅠ화XhoⅠ진행쌍매절화측서감정。중조재체전화대장간균E. coli BL21(DE3)균주,IPTG유도후,경SDS-PAGE전영분석화Western blot험증,순화TAT-IhC-Der p 1-3T단백후진행IgE결합시험。결과쌍매절화측서결과표명,성공구건료pET-28a-TAT-IhC-Der p 1-3T중조원핵표체재체;SDS-PAGE전영분석현시TAT-IhC-Der p 1-3T가유도표체;Western blot검측표명해융합단백순화성공;IgE결합시험표명TAT-IhC-Der p 1-3T결합옥진만과민병인혈청IgE적능력강우Der p 1변응원(P<0.001)。결론성공구건료가표체경MHC통로적편마Der p 1적3단T세포표위적중조pET-28a-TAT-IhC-Der p 1-3T재체,순화적TAT-IhC-Der p 1-3T구유교강적IgE결합능력,종이위후속경MHC통로적특이성면역치료전정기출。
Objective To construct a vector encoding T-cell epitopes of major allergen group 1 of Dermatophagoides pteronyssinus as a vaccine delivered by MHC class II pathway. Methods The nucleotide sequences of the 3 target genes were synthesized, including TAT, IhC and the recombinant fragment of Der p 1 encoding 3 T-cell epitopes. After amplification of the 3 target fragments by PCR and digestion with corresponding restriction endonucleases, the recombinant gene TAT-IhC-Der p 1-3T was ligated using T4 DNA ligase and inserted into the prokaryotic expression vector pET28a(+) to construct the recombinant plasmid pET-28a(+)-TAT-IhC-Der p 1-3T, which was confirmed by digestion with restriction endonucleases and sequencing. The recombinant vector was transformed into E. coli strain BL21 (DE3) and induced with IPTG, and the induced protein TAT-IhC-Der p 1-3T was detected by SDS-PAGE. After purification, the recombinant protein was confirmed by Western blotting and its allergenicity tested using IgE-binding assay. Results The recombinant plasmid pET-28a-TAT-IhC-Der p 1-3T was successfully constructed as confirmed by restriction endonuclease digestion and sequencing and the expression of the recombinant protein TAT-IhC-Der p 1-3T was induced in E. coli. Western blotting verified successfull purification of the target protein, which showed a stronger IgE-binding ability than Der p 1. Conclusion We successfully constructed a recombinant expression vector pET-28a-TAT-IhC-Der p 1-3T expressing a T-cell epitope vaccine delivered by MHC II pathway with strong IgE-binding ability, which provides a basis for further study on specific immunotherapy via MHC class II pathway.