猪2型圆环病毒核酸疫苗的接种Balb/c小鼠实验免疫研究

Experimental Study on Injecting Balb/c Mice with PCV2 Nucleotide Vaccines

为了筛选得到较理想的核酸疫苗,用pVAX1真核表达载体,以猪白介素18基因、猪2型圆环病毒内蒙株的衣壳蛋白和与复制相关蛋白基因为目的基因,构建了4个重组质粒,分别是pVAX1-ORF2、pVAX1-UL18ORF2、pVAX1-ORF2ORF1和pVAX1-IL18ORF2ORF1。用它们免疫6-8周龄Balb/c小鼠,进小鼠后一周首免,每隔19d免1次,共免3次;每10d采血1次,三免后10d杀鼠取脾。用ELISA方法检测小鼠血清抗体效价,流式细胞仪检测脾T淋巴细胞亚类CD4+、CD8+数量。统计学分析发现:4个核酸疫苗实验免疫组均是从最后一次采集的小鼠血清中,获得最高的抗体效价,与对照组PBS和空载体pVAX1相比都能产生一定的体液免疫反应,且以pVAx1-IL18ORF2ORF1核酸疫苗免疫小鼠产生的血清抗体效价最高;脾T淋巴细胞亚类数量测定发现,免疫实验组的CD4+与CD8+的T细胞亚类总数显著高于对照组(P<0.05),并以pVAX-1-IL18ORF2ORF1数值较高。结论是核酸疫苗pVAX1-IL18ORF2ORF1既能较好地刺激细胞免疫又能产生一定的体液免疫反应,相对较好,将作为候选核酸疫苗进行下一步本体动物免疫实验研究。

To select more ideal DNA vaccine, four recombinant plasmid were constructed as nucleotide vaccines, which were named as pVAX1-ORF2, PVAX1-ORF2ORF1, pVAX1-IL18ORF2 and pVAXl-IL18ORF2ORF1, respectively. Using pVAX1 as eukaryotic expressing vector, swine cytokine interleukin 18 gene and PCV2 (Innermongolia strain) capsid or replicon genes were aimed genes. Six/eight week-old Balb/c mices were divided into six groups, 12 mices each,two of which were control groups, inoculated by PBS and pVAX1 respectively,and the other four groups were for experimental use, injected by pVAXl-ORF2, pVAX1-ORF2ORF1, pVAX1-IL180RF2, pVAX1-IL18ORF2ORF1 ,respectively. It lasted one week until mice received the first intramuscular injection. Mice were inoculated three times, each every 20 days. Mice sera gained before inoculation was used as negative control. Then, during the immunization, mice sera were from the mice's tails every 10 days. On the 10th after the third inoculation, mice were killed by excising eyeballs and removing spleens with sterility. Detecting sera antibody using ELISA and calculating mice spleen T lymphocytes subgroups(CD4 + , CD8 + ) by flow cytometry. And find that the last collected sera has the highest litre among experimental groups; They all produced certain degree of humoral immunity compared with those from control groups, and the pVAXl-IL18ORF2ORF1 (naked DNA) is suprior. In addition, the numbers of experimental groups' CD4 + , CD8 + T lymphocytes subgroups are higher than the ones of control groups, the total number of T subgroups pVAXl-IL18ORF20RF1 group is also much higher. The result shows DNA vaccines named as pVAX1-IL18ORF20RF1 can efficiently stimulate both cellular immunity and humoral immunity. It will be tested continually as a specific PCV2 vaccine candidate