抗腮腺炎病毒抗体轻链基因的克隆及重组表达载体的构建

CLONING OF GENES ENCODING LIGHT CHAIN OF ANTIBODY AGAINST MUMPS VIRUSES AND CONSTRUCTION OF THE RECOMBINANT EXPRESSION VECTOR

目的 :构建抗腮腺炎病毒抗体轻链基因重组表达载体。方法 :从腮腺炎患者和腮腺炎抗体 Ig G阳性正常人群的淋巴细胞中提取总 RNA,逆转录成 c DNA。用相应的引物进行 PCR,扩增出轻链 κ和 λ基因 ,经 Sac I和 Xba I双酶切后 ,分别和噬粒载体 p Com b3连接 ,再经电穿孔转化大肠杆菌 XL1- blue菌株 ,将轻链基因克隆入载体 p Comb3。结果 :从分离出的淋巴细胞中共提取高质量 RNA约 110 μg,经逆转录、 PCR,分别扩增出约 70 0 bp大小的 κ和 λ基因。 PCR产物和载体经纯化、双酶切后进行连接 ,在 2 .5 kv,2 0 0 Ω,2 5 μF的条件下电转化 ,转化率为 :4 .14× 10 7,随机挑取 10个菌落进行 PCR鉴定 ,共有 6个克隆扩增出 κ基因 ,1个克隆扩增出 λ基因 ,轻链重组率为 70 %。结论 :获得的抗体轻链基因重组表达载体是高效、实用的 ,为下一步构建抗体 Fab段基因重组载体打下了良好的基础。

Objective:To construct the recombinant expression vector of genes encoding light chain of antibody against Mumps Viruses.Methods:The total RNA extracted from PBLs of patients and volunteers infected with Mumps Viruses was reverse transcripted,and light chain κ and λ genes of the Immunoglobulin were amplified by PCR using primers scanning κ and λ chain.After digestion with Sac I+Xba I,the amplified light chain fragments were cloned into phagemid pComb3 and electrotransinfected competent E.coli XL1-blue respectively.Results:Total 110μg RNA was extracted from PBLs with high quality.About 700bp κ and λ genes were amplified by RT-PCR respectively.The PCR products and pComb3 were ligated after purification and double digestion,and Electrotransinfection was conducted with 4.14×10 7 transforming ratio under the condition of 2.5kv,200Ω,25μF.PCR identification was conducted on 10 random clones.There were 6 clones which amplified κ genes,and 1 clone which amplified λ gene.The recombinant ratio of light chain was 70%.Conclusion:The recombinant expression vector bearing antibody light chain genes against Mumps Viruses is constructed effectively and practicably.Further construction of antibody Fab recombinant vector is undertaking.