B型肉毒神经毒素重链C-端片段的修饰及表达  

Modification and Expression of Gene Fragment at C-Terminus of Heavy Chain of Botulinum Neurotoxin Type B

作　　者：林琳[1] 林卫[2] 毛晓艳[2] 王棣[2] 

机构地区：[1]中华人民共和国江门出入境检验检疫局,江门529000 [2]兰州生物制品研究所第五研究室,兰州730046

出　　处：《中国生物制品学杂志》2009年第1期31-34,共4页Chinese Journal of Biologicals

摘　　要：目的修饰、克隆B型肉毒神经毒素重链C-端(BoNT/b HC)片段,并在大肠杆菌中进行表达。方法以B型肉毒梭状杆菌8806株基因组DNA为模板,PCR扩增B型肉毒神经毒素重链的转膜区和结合区,并以高频密码子替换BoNT/bHC基因N-端的5个低频密码子。将目的片段克隆入表达载体pET-42b,转化E.coli BL21(DE3)PlysS,IPTG诱导表达后,进行Western blot鉴定及可溶性分析。结果重组表达质粒经PCR及酶切鉴定证明构建正确;表达的重组蛋白相对分子质量约为80000,表达量占菌体总蛋白的32.6%,主要以包涵体形式存在;重组蛋白可被相应的抗BoNT/b全毒素的多克隆抗体所识别。结论已成功修饰并表达了B型肉毒神经毒素重链C-端片段,为进一步研制重组疫苗及高特异性的诊断试剂奠定了基础。Objective To modify and clone a gene fragment at C-terminus of heavy chain of botulinum neurotoxin （BoNT） type B, named as BoNT/b HC, and express in E. coli. Methods The BoNT/b HC gene encoding the transmcmbrane and binding domains of heavy chain of BoNT/b was amplified by PCR using the genomic DNA of strain 8806 of Clostridiurn botulinum type B as template, of which the five low frequency eodons at N-terminus were substituted with high frequency codons. The modified gene fragment was cloned into expression vector pET-42b, and the constructed recombinant plasmid was transformed to E. coli BL21 （DE3） PlysS for expression under induction of IPTG. The expressed product was identified by Western blot and analyzed for solubility. Resuits Both PCR and restriction analysis proved that recombinant plasmid pET-42b-rBoNT/b HC-TB was constructed correctly. The expressed recombinant protein with a relative molecular mass of about 80 000 mainly exised in a form of inclusion body, contained 32. 6% of total somatic protein and was recognized by polyclonal antibody against intact BoNT/b. Conclusion BoNT/b HC fragment was successfully modified and expressed, which laid a foundation of further development of recombinant vaccine and high specific diagnostic kit.

关 键 词：B型肉毒神经毒素 重链 C-端 修饰 表达 

分 类 号：R595.7[医药卫生—内科学] Q786[医药卫生—临床医学]