Click for new scientific resources and news about Corona[COVID-19]

Paper Information

Journal:   JOURNAL OF ADVANCES IN MEDICAL AND BIOMEDICAL RESEARCH   SUMMER 2007 , Volume 15 , Number 59; Page(s) 21 To 34.
 
Paper: 

IMMUNOLOGICAL CHARACTERIZATION OF LIGHT CHAIN RECOMBINANT PROTEIN OF CLOSTRIDIUM BOTULINUM TYPE A

 
 
Author(s):  MOUSAVI S.L.*, AMANI J., NAZARIAN SH., SOROURI R.
 
* DEPARTMENT OF BIOLOGY, SHAHED UNIVERSITY, TEHRAN, IRAN
 
Abstract: 

Background and Objective: Botulinum neurotoxin type A, structurally consists of a 50KD light chain and a 100KD heavy chain linked by a disulfide bond. The protein can further be divided into three functional domains of which catalytic domain corresponds to the light chain. In this research we aimed to produce recombinant catalytic domain in order to obtain a protective protein.
Materials and Methods: Bacteria were grown in anaerobic conditions and genomic DNA was extracted by alkaline method. Following the gene coding a set of primers was designed and the catalytic domain was amplified through PCR. The PCR product was then cloned into three expression vectors namely pRSETA, pET28
a and pET32a. The expressed protein was analyzed on SDS-PAGE and confirmed by ELISA and western blotting and then purified by affinity chromatography.
Results: In this research the maximum expression was obtained at 0.5 mM IPTG,OD600: 0.6 and 15 hours of induction at 30oC. The protein so expressed was purified by affinity coulmn chromatography The antibody raised against recombinant protein could protect the rats by 100 LD50 .
Discussion: Though the expression of AT- rich genes in E.coli system is low, we could obtain an appropriate level of expression in this study. The purification of recombinant protein in the early stages was elusive in the extreme by affinity chromatography due to the weak binding of histidine N-terminal to the column, howerer 90% purification was achieved through modification of the technique. The antibody produced against this domain was less protective compared to that of binding domain.

 
Keyword(s): CLOSTRIDIUM BOTULINUM TYPE A, CATALYTIC DOMAIN, PCR, RECOMBINANT PROTEIN
 
References: 
  • ندارد
 
  Persian Abstract Yearly Visit 74
 
Latest on Blog
Enter SID Blog