Enhanced expression of recombinant beta toxin of Clostridium perfringens type B using a commercially available Escherichia coli strain.

Fatemah Bakhshi, Reza Pilehchian Langroudi, Bahram Golestani Eimani

Overview

In the present study, recombinant plasmid pET22b? was transformed into E. coli strains BL21(DE3) and RosettaTM(DE3). The SDS-PSAGE analysis showed no CPB band from E. coli/BL21/ pET22? but showed a CPB band from E. coli/RosettaTM/pET22?. The sequencing analysis of purified pET22? revealed that the inserted gene had 987 bp, demonstrating a 99% – 100% identity with cpb genes that were previously deposited in GenBank. The recombinant toxin was expressed 30 min after induction with IPTG and continued for 18 h.

Summary

pET22b(+) and E. coli strain RosettaTM(DE3) are suitable expression vectors and hosts that can enhance the expression and production of C. perfringens recombinant beta toxin. Therefore, the E. coli/RosettaTM/pET22? clone could be used for further research on recombinant vaccine production.

Author Comments

Dr. Reza Pilehchian Langroudi, PhD
Dr. Reza Pilehchian Langroudi, PhD
Razi vaccine and serum research institute
Molecular Genetics
Karaj, Alborz | Iran (Islamic Republic of)
Iranian variant type B was isolated in 1954 from intestinal contents of sheep and goats. The three strains of Clostridium welchii type B isolated were different from the classical typeB strains in their production of kappa and non-production of lambda and hyaluronidase toxins. Two of the strains were isolated from young goats and the other from an adult sheepDr. Reza Pilehchian Langroudi, PhD

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Enhanced expression of recombinant beta toxin of Clostridium perfringens type B using a commercially available Escherichia coli strain.

Authors:
Dr. Reza Pilehchian Langroudi, PhD
Dr. Reza Pilehchian Langroudi, PhD
Razi vaccine and serum research institute
Molecular Genetics
Karaj, Alborz | Iran (Islamic Republic of)

Onderstepoort J Vet Res 2016 Jun 30;83(1):a1136. Epub 2016 Jun 30.

Razi Vaccine and Serum Research Institute, Agricultural Research, Education and Extension Organization (AREEO), Alborz, Karaj.

Clostridium perfringens beta toxin is only produced by types B and C and plays an important role in many human and animal diseases, causing fatal conditions that originate in the intestines. We compared the expression of C. perfringens type B vaccine strain recombinant beta toxin gene in the Escherichia coli strains Rosetta(DE3) and BL21(DE3). The beta toxin gene was extracted from pJETβ and ligated with pET22b(+). pET22β was transformed into E. coli strains BL21(DE3) and Rosetta(DE3). Recombinant protein was expressed as a soluble protein after isopropyl β-D-1-thiogalactopyranoside (IPTG) induction in strain Rosetta(DE3) but not in BL21(DE3). Expression was optimised by growing recombinant cells at 37 °C and at an induction of 0.5 mM, 1 mM, 1.5 mM IPTG. Expression was evaluated using sodium dodecyl sulfate Polyacrylamide gel electrophoresis (SDS-PAGE). The recombinant protein was purified via Ni-NTA and was analysed using western blot. We concluded that E. coli strain RosettaTM(DE3) can enhance the expression of C. perfringens recombinant beta toxin.

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Source
http://dx.doi.org/10.4102/ojvr.v83i1.1136DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6238768PMC
June 2016
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References

(Supplied by CrossRef)

Brooks et al.
British Veterinary Journal 1957

Cavalcanti et al.
RBCF Journal of Pharmaceutical Sciences 2004

Hunter et al.
Infectand Immunity 1993

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