CAJ | 학술논문

目的利用重组BCG (rBCG)技术,构建能以串联和并联形式、在细菌表面表达Der p2和Omp D抗原的两种rBCG 口服疫苗.方法经PCR法分别扩增获得Der p2和Omp D基因,测序正确后将两者克隆入原核表达载体pProEX HTb,获得pProEX HTb Der p2-Omp D质粒.①串联表达:将Der p2-Omp D融合基因亚克隆入穿梭胞壁表达载体pCW,经酶切鉴定阳性命名为pCW Der p2-Omp D 质粒.将此重组质粒电穿导入BCG感受态细胞,构建可胞壁串联表达Der p2-Omp D融合蛋白的rBCG；②并联表达:构建pCW-Der p2与pCW-Omp D两种质粒,并将二者同时电穿导入BCG感受态细胞,以构建胞壁并联表达Der p2和Omp D蛋白的rBCG.经潮霉素抗性筛选的阳性克隆,均采用以下三种方式进行鉴定:PCR特异性扩增目的基因片段；用兔抗Der p2多克隆抗体与兔抗Omp D多克隆抗体对阳性克隆分别进行斑点免疫杂交法和间接免疫荧光法鉴定.结果采用基因工程手段制备出以胞壁形式串联和并联表达Der p2和Omp D蛋白的两种rBCG口服疫苗,并通过PCR、斑点免疫杂交法及间接免疫荧光法分别进行鉴定.结论两种rBCG 口服疫苗构建成功,为体外实验和临床应用提供基础.
목적 이용중조BCG (rBCG)기술,구건능이천련화병련형식、재세균표면표체Der p2화Omp D항원적량충rBCG 구복역묘.방법 경PCR법분별확증획득Der p2화Omp D기인,측서정학후장량자극륭입원핵표체재체pProEX HTb,획득pProEX HTb Der p2-Omp D질립.①천련표체:장Der p2-Omp D융합기인아극륭입천사포벽표체재체pCW,경매절감정양성명명위pCW Der p2-Omp D 질립.장차중조질립전천도입BCG감수태세포,구건가포벽천련표체Der p2-Omp D융합단백적rBCG；②병련표체:구건pCW-Der p2여pCW-Omp D량충질립,병장이자동시전천도입BCG감수태세포,이구건포벽병련표체Der p2화Omp D단백적rBCG.경조매소항성사선적양성극륭,균채용이하삼충방식진행감정:PCR특이성확증목적기인편단；용토항Der p2다극륭항체여토항Omp D다극륭항체대양성극륭분별진행반점면역잡교법화간접면역형광법감정.결과 채용기인공정수단제비출이포벽형식천련화병련표체Der p2화Omp D단백적량충rBCG구복역묘,병통과PCR、반점면역잡교법급간 접면역형광법분별진행감정.결론 량충rBCG 구복역묘구건성공,위체외실험화림상응용제공기출.
Objective To construct two oral rBCG vaccines expressing Der p2 and Omp D (in form of series connection and parallel connection) antigens on its cell wall.Methods The genes of Der p2 and Omp D were first amplified by PCR,and then were ligated into pProEX HTb vector respectively,and gained Der p2-Omp D fused gene.①Expressing in form of series connection:The Der p2-Omp D fused gene was subcloned into the shuttle plasmid pCW.The positive recombinant plasmid was named pCW Der p2-Omp D,and was transformed into BCG competent cells to construct rBCG oral vaccine,which could express the Der p2-Omp D fused protein on its cell wall in the form of series connection.②Expressing in form of parallel connection:The pCW-Der p2 and pCW-Omp D plasmids were transformed into BCG competent cells together to construct the another rBCG oral vaccine,which could express Der p2 and Omp D proteins on its cell wall in the form of parallel connection.The two rBCG oral vaccines were selected by hygromycine,and the positive rBCG were identified by PCR,spot immunifaction and indirect immunofluorescence.The PCR result showed that there was specific amplification of the target gene.Spot immunifaction showed that specific banding of rBCG expressing product with Omp D pAb and Der p2 pAb was observed,indicating that rBCG possesses immunological function.Indirect immunofluorescence showed that specifc green immunofluorescences were observed,indicating that rBCG could express the goal protein on its surface.Results The two rBCG oral vaccines expressing Der p2 and Omp D proteins on its cell wall in the forms of series connection and parallel connection were preparated by genetic engineering,and then identified by PCR,spot immunifaction and indirect immunofluorescence successfully.Conclusions Two rBCG oral vaccines were constructed successfully,which has provided foundation for in vitro experimental and clinical application.