摘要
为掌握猪圆环病毒2型(PCV2)ORF3蛋白免疫小鼠后引起其细胞因子变化的特征,设计了特异性引物,以PCV2FJ948167毒株的基因组DNA为模板,PCR扩增获得ORF3基因,并将其克隆到pSMK-sumo原核表达载体。把构建成功的重组质粒转化至BL21(DE3RIL strain)表达菌株,IPTG诱导后SDS-PAGE和Western-blot分析显示,ORF3基因已在大肠杆菌中表达,表达产物的分子质量约为32ku。将分离纯化后的ORF3蛋白免疫小鼠,检测免疫后不同时间点IFN-γ、IL-4、IL-10和IL-12在血清中的质量浓度。结果显示,免疫后小鼠血清中IL-4的质量浓度显著升高(P<0.05),而IL-10、IL-12和INF-γ的质量浓度没有明显的变化。结果表明,ORF3蛋白在PCV2诱发的体液免疫中发挥一定的作用。
To research the profiles changes of cytokine in the immunized mice with porcine circovirus type 2（PCV2） ORF3 protein,the specific primers were designed according to the published sequences of ORF3 gene of PCV2. The complete DNA fragment encoding ORF3 protein was obtained from viral DNA of PCV2 FJ948167 strain by PCR and the ORF3 gene was cloned into the prokaryotic expression vector pSMK-sumo. The resultant recombinant plasmid was named as ps-ORF3;and it was then transformed into BL21（DE3 RIL strain）. SDS-PAGE and Western-blot analyses showed that the aim protein was expressed when it was induced by 0.5 mmol/L IPTG. The recombinant ORF3 protein was soluble and in molecular mass of 32 ku. Mice were vaccinated with the purified recombinant protein. The concentrations of IFN-γ, IL-4,IL-10 and IL-12 in the peripheral blood were detected by a sandwich ELISA. In result,when compared with the control group,the content of IL-4 was increased significantly,but the content of IFN-γ,IL-10 and IL-12 has no significant changes. The result suggested that ORF3 protein plays a certain role in the humoral immunity in mice.
出处
《中国兽医科学》
CAS
CSCD
北大核心
2013年第6期583-587,共5页
Chinese Veterinary Science
