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

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MOUSAVI S.L.; AMANI J.; NAZARIAN SH.; SOROURI R.

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Journal: Journal of Advances in Medical and Biomedical Research Year:2007 | Volume:15 | Issue:59 Page(s): 21-34

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Title

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

Author(s)

MOUSAVI S.L. | AMANI J. | NAZARIAN SH. | SOROURI R. | Issue Writer Certificate

Keywords

CLOSTRIDIUM BOTULINUM TYPE AQ2

CATALYTIC DOMAINQ3

PCRQ2

RECOMBINANT PROTEINQ2

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, pET28a 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.

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References

CLONING, EXPRESSION AND PURIFICATION OF THE GENE CODING TRANSLOCATING DOMAIN OF BOTULINUM NEUROTOXIN TYPE A

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APA: Copy

MOUSAVI, S.L., AMANI, J., NAZARIAN, SH., & SOROURI, R.. (2007). IMMUNOLOGICAL CHARACTERIZATION OF LIGHT CHAIN RECOMBINANT PROTEIN OF CLOSTRIDIUM BOTULINUM TYPE A. JOURNAL OF ADVANCES IN MEDICAL AND BIOMEDICAL RESEARCH, 15(59), 21-34. SID. https://sid.ir/paper/61412/en

Vancouver: Copy

MOUSAVI S.L., AMANI J., NAZARIAN SH., SOROURI R.. IMMUNOLOGICAL CHARACTERIZATION OF LIGHT CHAIN RECOMBINANT PROTEIN OF CLOSTRIDIUM BOTULINUM TYPE A. JOURNAL OF ADVANCES IN MEDICAL AND BIOMEDICAL RESEARCH[Internet]. 2007;15(59):21-34. Available from: https://sid.ir/paper/61412/en

IEEE: Copy

S.L. MOUSAVI, J. AMANI, SH. NAZARIAN, and R. SOROURI, “IMMUNOLOGICAL CHARACTERIZATION OF LIGHT CHAIN RECOMBINANT PROTEIN OF CLOSTRIDIUM BOTULINUM TYPE A,” JOURNAL OF ADVANCES IN MEDICAL AND BIOMEDICAL RESEARCH, vol. 15, no. 59, pp. 21–34, 2007, [Online]. Available: https://sid.ir/paper/61412/en

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