■由结核分枝杆菌引起的结核病(M.结核病),仍然是全球死亡的主要原因之一,每年有近200万例死亡病例。卡介苗(BacilleCalmette-Guerin)仍然是世界上使用最广泛的疫苗,但是世界不同地区的保护性免疫不同。因此,包括DNA疫苗在内的新策略是必要的。本研究旨在设计并构建含有结核分枝杆菌mpt64基因的克隆载体。
■M.结核H37Rv在LowensteinJensen培养基上培养,提取基因组DNA。使用设计的特异性引物通过PCR扩增mpt64基因。用BamHI和EcoRI限制酶消化mpt64和pcDNA3.1(+)后,使用T4DNA连接酶将mpt64片段连接到消化的载体中。然后,将重组载体转化到感受态大肠杆菌(E.大肠杆菌)TOP10菌株。为了确认转化细菌的菌落,抗生素耐药性,菌落-PCR,使用限制性内切酶消化和DNA测序。
■要确认克隆,使用mpt64特异性引物进行菌落PCR,并通过凝胶电泳观察到718bp的片段。还通过使用BamHI和EcoRI限制酶的限制酶消化来验证克隆,并且观察到718bp片段。此外,DNA测序结果显示与GenBank中H37Rv的mpt64片段具有100%的同源性。
■在这项研究中,mpt64片段成功克隆到pcDNA3.1(+)载体中。该构建体可在未来的研究中用作动物模型中的DNA疫苗以诱导免疫系统应答。
UNASSIGNED: Tuberculosis caused by Mycobacterium tuberculosis (M. tuberculosis), remains as one of the leading causes of deaths worldwide, with nearly two million death cases annually. BCG (Bacille Calmette-Guerin) continues to be the most widely used vaccine in the world, but the protective immunity differs in different parts of the world. Accordingly, new strategies including DNA vaccines are essentially needed. This study was aimed to design and construct a cloning vector containing mpt64 gene of M. tuberculosis.
UNASSIGNED: M. tuberculosis H37Rv was cultured on Lowenstein Jensen medium, and genomic DNA was extracted. The mpt64 gene was amplified by PCR using designed specific primers. After the digestion of mpt64 and pcDNA3.1 (+) by BamHI and EcoRI restriction enzymes, the mpt64 fragment was ligated into the digested vector using T4 DNA ligase enzyme. Then, the recombinant vector was transformed into competent Escherichia coli (E. coli) TOP10 strain. To confirm the colonies of transformed bacteria, antibiotic resistance, colony-PCR, restriction enzyme digestion and DNA sequencing were used.
UNASSIGNED: To confirm the clones, colony-PCR using mpt64 specific primers was performed and the fragment of 718 bp was observed by gel electrophoresis. Clones were also verified by restriction enzyme digestion using BamHI and EcoRI restriction enzymes and the 718 bp fragment was observed. Furthermore, results of DNA sequencing showed 100% homology with the mpt64 fragment of H37Rv in GenBank.
UNASSIGNED: In this study, the mpt64 fragment was successfully cloned in pcDNA3.1 (+) vector. This construct can be used in future studies as a DNA vaccine in animal models to induce immune system responses.