Haghighat Setareh, Siadat Seyed Davar, Sorkhabadi Seyed Mehdi Rezayat, Sepahi Abbas Akhavan, Mahdavi Mehdi
Department of Biology, Faculty of Basic Sciences, Science and Research branch, Islamic Azad University, Tehran, Iran.
Avicenna J Med Biotechnol. 2013 Oct;5(4):204-11.
Staphylococcus aureus (S. aureus) is a major nosocomial pathogen and the infection with this organism in human is increasing due to the spread of antibiotic resistant strains. One of the resistance mechanisms of S. aureus comprises modification in binding proteins to penicillin. Vaccine strategy may be useful in controlling the infections induced by this organism. This study aimed at developing and producing the recombinant protein PBP2a as a vaccine candidate and evaluating the related humoral immune response in a murine model.
A 242 bp fragment of mecA gene was amplified by PCR from S. aureus COL strain and then cloned into prokaryotic expression vector pET-24a. For expression of recombinant protein, pET24a-mec plasmid was transformed into competent E. coli BL21 (DE3) cells. Recombinant protein was over expressed with 1 mM isopropythio-β-D-galctoside (IPTG) and purified using Ni-NTA agarose. SDS-PAGE and western blotting were carried out to confirm protein expression. For immunization of experimental groups, Balb/c mice were injected subcutaneously with 20 µg of recombinant PBP2a three times with three weeks intervals. The sera of experimental groups were collected three weeks after the last immunization and then specific antibodies were evaluated by ELISA method.
Successful cloning of mecA was confirmed by colony-PCR, enzymatic digestion, and sequencing. SDS-PAGE and western blot analysis showed that recombinant protein with molecular weight of 13 kDa is over expressed. In addition, high titer of specific antibody against PBP2a in vaccinated mice was developed as compared with the control group and confirmed the immunogenicity of the vaccine candidate.
Results suggest that PBP2a recombinant induced specific antibodies and can be used as Staphylococcal vaccine candidate after further studies.
金黄色葡萄球菌是一种主要的医院病原体，由于抗生素耐药菌株的传播，人类感染这种微生物的情况正在增加。金黄色葡萄球菌的耐药机制之一包括青霉素结合蛋白的修饰。疫苗策略可能有助于控制由这种微生物引起的感染。本研究旨在开发和生产重组蛋白PBP2a作为候选疫苗，并在小鼠模型中评估相关的体液免疫反应。
通过PCR从金黄色葡萄球菌COL菌株中扩增出mecA基因的242bp片段，然后克隆到原核表达载体pET-24a中。为了表达重组蛋白，将pET24a-mec质粒转化到感受态大肠杆菌BL21(DE3)细胞中。用1mM异丙基硫代-β-D-半乳糖苷(IPTG)过量表达重组蛋白，并用Ni-NTA琼脂糖进行纯化。进行SDS-PAGE和western印迹以确认蛋白表达。对于实验组的免疫，将Balb/c小鼠皮下注射20μg重组PBP2a，共注射三次，间隔三周。在最后一次免疫后三周收集实验组的血清，然后通过ELISA方法评估特异性抗体。
通过菌落PCR、酶切和测序证实了mecA的成功克隆。SDS-PAGE和western印迹分析表明，分子量为13kDa的重组蛋白过量表达。此外，与对照组相比，接种疫苗的小鼠中产生了高滴度的抗PBP2a特异性抗体，证实了候选疫苗的免疫原性。
结果表明，PBP2a重组体诱导了特异性抗体，经过进一步研究后可作为葡萄球菌疫苗候选物。
