重组嵌合抗人CD22四价基因工程抗体在毕赤酵母中的表达及活性检测

Optimal expression and activity detection of recombinant,chimeric and genetically engineered tetravalent anti-human CD22 antibodies in P.pastoris

目的:采用酵母多拷贝分泌表达载体pPIC9K表达重组嵌合抗人CD22四价基因工程抗体cRFB4FC和cRFB4CH3,并进行活性检测。方法:采用基因克隆技术获得两抗体的基因,然后将其克隆入表达载体pPIC9K中,构建重组质粒pPIC9K/cRFB4FC和pPIC9K/cRFB4CH3,经测序鉴定正确,电转化入毕赤酵母SMD1163中,将经G418抗性筛选出的重组酵母菌株进行PCR鉴定,经甲醇诱导,表达产物经SDS-PAGE分析和Western blot鉴定,经ELISA检测生物学活性。采用正交试验优化重组酵母菌株的表达条件以提高抗体的表达量。结果:获得重组表达质粒pPIC9K/cRFB4FC和pPIC9K/cRFB4CH3,经G418筛选及PCR鉴定得到高拷贝整合的重组酵母菌株,经甲醇诱导表达出cRFB4FC和cRFB4CH3蛋白。它们在SDS-PAGE上分别表现为相对分子质量(Mr)约326 000和295 000的蛋白区带,在Western blot分析中均可被山羊抗人IgG Fc单克隆抗体(mAb)识别,经ELISA分析它们都具有较高的生物学活性。优化表达条件后,抗体的表达量分别提高了196.4%和151.7%。结论:抗体cRFB4FC和cRFB4CH3在酵母菌SMD1163中成功获得高效分泌表达,并有免疫学活性。

AIM： To express the recombinant, chimeric and genetically engineered tetravalent anti-human CD22 anti- bodies （cRFB4FC and cRFB4CH3） using yeast cells secreting type carrier pPIC9K, and screen for optimal conditions of engineered yeast cells expressing cRFB4FC and cRFEMCH3. METHODS： The genes of cRFB4FC and cRFB4CH3 were cloned into P. pastoris eukaryotic expres- sion vector pPIC9K to construct the recombinant plasmids pPIC9K-cRFB4FC and pPICgK-cRFB4CH3, then identified by DNA sequencing. The recombinant plasmid pPICgK- cRFB4FC and pPIC9K-cRFB4CH3 were transfected into P. pastoris SMD]]63. The recombinant yeast cell line cho- sen by G418 resistance was identified by PCR. After metha- nol induction, the expressed protein products were verified by SDS-PAGE and Western blotting, and biologic activity was identified by ELISA. Finally, orthogonal test was con- ducted to optimize the expression conditions of the recombi- nant yeast cell lines so as to increase the expression level of the antibodies. SIJLTS： The secretory type yeast carriers pPICgK-cRFB4FC and pPICgK-cRFB4CH3 were successful- ly constructed and chosen by G418 as well as identified by PCR to obtain the highly copied and integrated recombinant yeast cell line. The cRFB4FC and cRFB4CH3 proteins were expressed by yeast cells containing pPICgK-cRFB4FC and pPICgK-cRFB4CH3 induced by methanol. The relative molecular mass （Mr） of cRFB4FC and cRFB4CH3 were about 326 000 and 295 000 Da. They could be identified by goat anti-human IgG （ Fc ） monoclonal antibody with Western blotting on SDS-PAGE, and higher biologic activity was confirmed by ELISA. Under the optimized expression conditions, the mean expression levels of cRFB4FC and cRFB4CH3 in recombinant yeast increased by 196.4% and 151.7%. CONCLUSION： The recombinant, chimeric and genetically engineered tetravalent anti-human CD22 antibodies （cRFB4FC and cRFB4CH3 ） proteins can be highly expressed in the P. pastoris SMD1163 expression system, and possess immunological activity.