ISSN: 0973-7510

E-ISSN: 2581-690X

Lei Yang, Xiangguo Wang, Yujie Song, Qian Li , Rui Dai, Guangdong Zhang, Yaping Jin and Aihua Wanga
1Key Laboratory of Animal Biotechnology, Ministry of Agriculture, PR China College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, 712100, P.R. China.
J Pure Appl Microbiol. 2014;8(5):4045-4050
© The Author(s). 2014
Received: 18/02/2014 | Accepted: 21/04/2014 | Published: 31/10/2014
Abstract

To clone the Omp16, Omp19 and Omp28 genes in pET32a expression vector and purify recombinant proteins used as antigen for future serological test development. Brucella melitensis S19 strain was cultured and bacterial DNA was extracted. Oligonucleotide primer pair was designed based on Genbank gene sequence with EcoR I and Sal I restriction site at 5´end of the forward and reverse primers respectively. DNA amplification was performed using PrimSTAR HS DNA polymerase and the PCR product was purified by Tiangen DNA Purification Kit. Purified DNA was excised with EcoR I and Sal I from the PCR product and subsequently cloned into pET32a. Target protein expression was induced by adding IPTG to a ûnal concentration of 1 mM when the culture OD600 (optical density at 600 nm) reached 0.6. targeted proteins wereobtained using Ni-NTA agarose resin. Brucella Omp16, Omp19 and Omp28 genes was successfully cloned into pET32a vector. Fusion proteins were expressed and puriûed. The Omp16 Omp19 Omp28 genes were cloned into pET32a and fusion proteins were purified.

Keywords

Omp16, Omp19, Brucella, DNA

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