犬Ⅰ型腺病毒结构蛋白Knob的可溶性表达及免疫原性研究

Study on the soluble expression and immunogenicity of structural protein Knob of Canine adenovirus type 1

为了探究犬Ⅰ型腺病毒(Canineadenovirus type1,CAdV-1)F1301株结构蛋白Knob的免疫原性,试验采用原核表达技术将Knob蛋白基因克隆到pColdⅡ原核表达载体中进行低温诱导,并在大肠杆菌BL21(DE3)感受态细胞中实现可溶性表达。利用纯化后的重组蛋白Knob免疫Balb/c小鼠,监测免疫后小鼠血清中重组蛋白Knob特异性抗体ELISA效价、病毒中和抗体效价,测定脾脏T淋巴细胞增殖活性,研究重组蛋白Knob的免疫原性。结果表明:表达的重组蛋白Knob为可溶性蛋白,分子质量约为20ku。第2次免疫后7,14天,试验组小鼠血清ELISA抗体效价分别为1∶6400和1∶3200,中和抗体效价分别为1∶118和1∶96。第2次免疫后7天,试验组小鼠T淋巴细胞增殖指数为1.322,显著高于对照组(P<0.05)。说明试验成功表达出CAdV-1重组蛋白Knob,所表达的重组蛋白Knob具有良好的免疫原性且存在CAdV-1的中和抗原表位。

In order to explore the immunogenicity of structural protein Knob of Canine adenovirus type 1 ( CAdV-1) F1301 strain,prokaryotic expression method was adopted in this experiment for the cloning of Knob protein into pColdⅡexpression vector,which was induced under low temperature. Soluble expression was achieved in Escherichia coli BL21 ( DE3) competent cells. The purified recombination protein Knob was immunized to the Balb/c mice. After immunization,specific antibody ELISA titers and virus neutralizing antibody titers against Knob protein in mouse sera were monitored and the spleen T lymphocyte proliferation activity was determined,which was for the study of the immunogenicity of recombinant protein Knob. The results showed that the expressed recombinant protein Knob was a soluble protein with a molecular weight of about 20 ku. Mouse serum ELISA titers of experimental group were 1 ∶6 400 and 1 ∶3 200 on 7 and 14 days post the second immunization respectively,and the neutralizing antibody titers were 1 ∶118 and 1 ∶96 respectively. On 7 days post the second immunization,the mouse T lymphocyte proliferation index of experimental group was 1. 322,which was significantly higher than that of control group( P<0. 05). The results suggested that the recombinant protein Knob of CAdV-1 was successfully expressed,which had good immunogenicity and neutralizing antigenic epitopes of CAdV-1.