A型肉毒毒素保护性抗原的基因修饰及高效表达

Modification and High Level Expression of Protective Antigen Fragment of Botulinum Neurotoxin Serotype A

在A型肉毒毒素保护性抗原基因初步表达的基础上 ,为提高表达水平 ,依据EMBL的DNA数据库中A型肉毒毒素基因全序列 ,重新设计上游引物 ,通过修饰基因片段N端 ,保持氨基酸序列不变 ,从已获得的A型肉毒毒素与靶细胞起结合作用的重链C端基因中 ,扩增小的突变基因 ,克隆入pGEM T载体进行测序 ,并以pBV2 2 0为表达载体构建重组表达质粒 ,在大肠杆菌中实现高效表达。结果表明 ,重组表达产物占全菌蛋白的 4 0 % ,酶联检测重组表达产物具有特异结合活性。A型肉毒毒素保护性抗原基因的高效表达 。

Designed a pair of primers through modifying N terminal bases(5bps) of gene after ATG but not changing amino acid, and amplified a smaller mutated gene sequnce (468bp) containing two protective antigenic determinants from pBlue BoNTaHc, N terminal codon of mutated gene fragment is changed from low to high frenqence in E.coli . Mutated gene was ligated into pGEM T vector and sequenced, then, cloned into a expression plasmid pBV220 As a result, cloned gene was expressed in insoluble form by temperature inducing (from 30℃to 42℃)in E.coli ,.Expression pruduct is 40% of total proteins and is of specific binding activity to antibody in ELISA.The successful modification and high level expression of protective fragment of botulinum neurotoxin serotype A(BoNTaHc468) gene is conducive to further study on antitoxin and vaccine.