乙型肝炎病毒前S1、前S2和S抗原重组毕赤酵母表达菌株的基因和表达稳定性

Genetic and Expression Stability of Recombinant Pichia pastoris Expressing Hepatitis B Surface Antigen Containing PreS1,PreS2 and S Epitopes

目的研究同时表达SS1、SS2两种多肽(含前S1、前S2和S三种抗原成分)的重组毕赤酵母工程菌株(GS115-SS1S2)基因和表达的稳定性。方法取工程菌原代种子,连续传代至30代,提取15和30代工程菌基因组DNA,PCR分别扩增SS1和SS2基因片段,克隆到pBluescriptⅡSK(+)载体中,进行基因序列测定。对第5、10、15、20、25、30代菌种进行培养和诱导,制备抗原粗提液,并用ELISA法检测其中的前S1、前S2和S的抗原性,同时用RPHA法检测抗原效价。结果第15和30代菌种基因组中SS1和SS2基因序列与原始菌种完全一致,第5、10、15、20、25、30代菌种表达产物的前S1、前S2和S抗原均为阳性;菌种传至20代,抗原滴度无变化,25代和30代菌种抗原滴度有一定程度下降。结论重组毕赤酵母工程菌GS115-SS1S2在20代传代过程中,基因和表达稳定性良好。

Objective To study the genetic and expression stability of a recombinant Pichia pastoris strain （GS115-SS1S2） expressing hepatitis B surface antigen （HBsAg） containing preS1, preS2 and S epitopes. Methods Subculture primary GS115-SS1S2 strain continuously for 30 passages. Extract genomic DNA from the strain of 15 and 30 passages for amplification of SS1 and SS2 gene fragments by PCR. Clone the amplified gene fragments to vector pBluescript II SK（ ＋ ）. Transform the constructed recombinant plasmid to E. coli DH5α, and screen positive clones for restriction analysis and DNA sequencing. The recombinant strains of passages 5, 10, 15, 20, 25 and 30 were cultured and induced with methanol, and the cultures were collected to prepare crude antigen extract in which preS1, preS2 and S antigens were determined by ELISA and the antigen titer by RPHA. Results Both the SS1 and SS2 gene sequences in genomes of recombinant strain of passages 15 and 30 were completely consistent with those of primary strain. The expressed products of recombinant strains of passages 5, 10, 15, 20, 25 and 30 were positive for preS1, preS2 and S antigens. Compared with that of primary strain, the antigen titer of expressed product of recombinant strain of passage 20 showed no significant difference, while those of passages 25 and 30 showed a certain decrease. Conclusion Recombinant Pichia pastoris strain GS115-SS1S2 within 20 passages showed good genetic and expression stabilities during subculture.