布鲁氏菌 16M 株 virB12 基因在 pET28a 载体中的分子克隆。

Molecular cloning of virB12 gene of Brucella melitensis 16M strain in pET28a vector.

机构信息

Tehran University of Medical Sciences, School of Medicine, Microbiology Department, Tehran, Iran.

出版信息

Asian Pac J Trop Med. 2012 Jul;5(7):511-3. doi: 10.1016/S1995-7645(12)60089-3.

DOI:10.1016/S1995-7645(12)60089-3

PMID:22647810

Abstract

OBJECTIVE

To clone the virB12 gene in pET28a expression vector for production of recombinant protein to be used as antigenic component for future serological test development.

METHODS

Brucella melitensis (B. melitensis) 16M strain was cultured and bacterial DNA was extracted by Bioneer AccuPrep® Genomic DNA Extraction Kit. Oligonucleotide primer pair was designed based on Brucella virB12 gene sequence with BamHI and HindIII 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 DNA AccuPrep®Gel Purification Kit. Purified DNA was cloned into pJET1.2 cloning vector. VirB12 gene fragment was excised from pJET1.2 using BamHI/HindIII and subsequently subcloned into pET28a (+).

RESULTS

Brucella virB12 gene was successfully cloned in pJET1.2 and then in pET28a (+) plasmids. PCR and restriction enzyme digestion confirms the procedure.

CONCLUSION

We cloned and expressed the Brucella virB12 gene which could be used as antigenic component for specific serological assay development.

摘要

目的

将 virB12 基因克隆到 pET28a 表达载体中，用于生产重组蛋白，作为未来血清学检测开发的抗原成分。

方法

培养布鲁氏菌（B. melitensis）16M 株，使用 Bioneer AccuPrep®基因组 DNA 提取试剂盒提取细菌 DNA。根据布鲁氏菌 virB12 基因序列设计寡核苷酸引物对，正向引物和反向引物的 5'端分别带有 BamHI 和 HindIII 限制性酶切位点。使用 PrimSTAR® HS DNA 聚合酶进行 DNA 扩增，使用 DNA AccuPrep®Gel Purification Kit 纯化 PCR 产物。纯化的 DNA 克隆到 pJET1.2 克隆载体中。从 pJET1.2 中用 BamHI/HindIII 酶切切下 virB12 基因片段，然后亚克隆到 pET28a（+）中。