重组质粒βhCG-pPIC9K的构建及嗜甲醇酵母(Pichiapastoris)的转化  

Construction of recombination plasmid βhCG -pPIC9K and transformation into Pichia pastoris

作　　者：陈晓丽[1] 潘善培[1] 芦春斌[1] 谢琪璇[1] 肖銮娟[1] 

机构地区：[1]暨南大学生殖免疫研究中心,广东广州510632

出　　处：《暨南大学学报（自然科学与医学版）》2000年第5期116-121,共6页Journal of Jinan University(Natural Science & Medicine Edition)

基　　金：广东省自然科学基金!(940 36 5 )

摘　　要：目的 :为用基因重组技术表达hCG避孕疫苗抗原 ,构建在酵母细胞中表达的重组质粒βhCG -pPIC9K ,转化嗜甲醇酵母 .方法 :根据βhCG的cDNA序列设计两条引物 ,使上游带EcoRI酶切位点 ,下游带NotI酶切位点 ,以质粒 βhCG -PBSKS为模板 ,进行PCR扩增反应 ;将所得的DNA片段经EcoRI和NotI双酶切后用T4连接酶与pPIC9K质粒进行连接 ,然后导入大肠杆菌DH5α ,用PCR筛选阳性克隆并用双酶切鉴定重组子βhCG -pPIC9K .再用电打孔法转化酵母 (Pichiapastoris) ,用缺组氨酸MD平板筛选阳性株并在含G4 18的YPD培养基中筛选多拷贝插入菌株 .结果 :用所设计的引物扩增出两端分别带EcoRI和NotI酶切位点的βhCGDNA片段 ,插入pPIC9K并导入DH5α获得阳性克隆 ,经PCR反应和双酶切鉴定表明重组质粒是 βhCG -pPIC9K ,并转化嗜甲醇酵母获得耐受 4mg/mLG4 18的菌株 .结论 :构建了重组质粒 βhCG -pPIC9K并获得插入重组质粒 βhCGAim: To construct a recombinant plasmid β-hCG-pPIC9K for expressing β-hCG antigen to develop contraceptive vaccine. Methods: β-hCG DNA fragment with EcoR I and Not I at 5' and 3' termini was prepared using plasmid β-hCG-PBS kS as template by PCR with a pair of designed primers which carried EcoR I in upstream and Not I in downstream. After digested by both EcoR I and Not I, β-hCG DNA fragment was linked with vector plasmid pPIC9K by T4 DNA ligase and was transformed into competent DH5 α. Positive clone was screened and plasmid β-hCG-pPIC9K was detected by PCR and digested with both EcoR I and Not I. Pichia pastoris was transformed with linearized β-hCG-pPIC9K by electroporation. The positive transformed strains were screened on MD plate. G418 resistant colonies with multicopy inserts were screened on YPD plates with increasing concentrations of G418. Results: β-hCG DNA fragment was inserted into vector pPIC9K. β-hCG DNA was linked with vector plasmid pPIC9K correctly. β-hCG-pPIC9K was transformed into Pichia pastoris. G418 resistant colonies with multicopy inserts was obtained. Conclusion: The recombinant plasmid β-hCG-pPIC9K was constructed. Pichia pastoris with inserted β-hCG-pPIC9K was obtained.[

关 键 词：重组质粒 嗜甲醇酵母 生殖免疫学 基因载体 

分 类 号：Q782[生物学—分子生物学]