为了建立一个简捷有效的抗血清的制备方法,本研究选用猪Ⅱ型圆环病毒核衣壳蛋白基因,使用水流动力学基因免疫的方法制备高效价抗血清的可行性.应用无内提取试剂盒制备猪Ⅱ型圆环病毒核衣壳蛋白基因真核表达载体pcDNA-Cap的无内毒素质粒.将该质粒使用水流动力学尾静脉注射法对小鼠(Mus musculus)进行基因免疫,重复免疫5次后采血收集血清;以原核表达获得的N末端去除了核定位序列的猪圆环病毒核衣壳蛋白表达产物作为抗原蛋白,以制备的小鼠血清作为一抗进行酶联免疫吸附试验(ELISA)和Western blot检测.结果显示,应用水流动力学尾静脉注射法获得抗血清稀释5000倍通过Western blot至少能够检测到10 ng的抗原蛋白,ELISA分析表明,其效价可达到1∶1000000,说明获得的抗血清具有很好的效价水平.这一研究为猪Ⅱ型圆环病毒相关研究用抗体的制备提供了一个简洁有效的方法,也为猪Ⅱ型圆环病毒的防治方法的建立提供了一个值得尝试的策略.%In order to establish a simple and efficient anti-serum preparation method for molecular biology research, in this research, porcine circovirus type II capsid protein gene was selected to research on the possiblity of preparing high titer anti-serum by hydrodynamics gentic immunization. Endotoxin free plasmid of pcDNA-Cap, with would express porcine circovirus type II capsid protein in the exkaryotic cell, was prepared using endotoxin free plasmid preparing kit and was delivered by hydrodynamics tail vein injection method for genetic immunization of mice (Mus musculus). After 5 times continuous immunization, the blood serum was collected. Using porcine circovirus type II capsid protein which has been deleted its nuclear laction signal sequence at its N-terminal and expressed in Escherichia coli as antigen, the prepared anti-serum was tested by enzymeliked immuno sorbent assay(ELISA) and Western blot. The results showed that the anti-serum prepared by hydrodynamics tail vein gene delivery method had very high titer(l: 1 000 000) by ELISA and could detect at least 10 ng antigen after 5 000-fold dilution in Western blotting analysis. These results showed that hydrodynamics gene delivery method may become an efficient method for preparing antibody which will be used in porcine circovirus type II related research. This study provides an alternative method for prevention the disease caused by porcine circovirus type II.
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