PCV2-HPS二联亚单位疫苗对小鼠免疫保护效果的评价

Evaluation of protective effect of porcine circovirus type 2-Haemophilus parasuis binary subunit vaccine on mice

为评价猪圆环病毒2型-副猪嗜血杆菌二联亚单位疫苗(以下简称二联苗)对小鼠的免疫保护效果,本研究将猪圆环病毒2型(PCV2)cap基因克隆至pMAL-c5X载体中,并通过原核系统表达了重组Cap蛋白(rCap)。通过western blot检测显示原核表达的rCap与PCV2单克隆抗体发生特异性结合,表明rCap具有良好的反应原性。利用本研究室已经纯化并保存的副猪嗜血杆菌(HPS)重组蛋白rCdtB、rAfua和rOPPA,与rCap蛋白以及佐剂ISA201VG混合乳化后制成PCV2-HPS二联亚单位疫苗,疫苗中各蛋白(rCdtB、rAfua、rOPPA、rCap)浓度均为50μg/300μL。将60只6周龄BALB/c小鼠随机均分成免疫组和对照组,采用背部多点注射方式免疫,首免后间隔14 d二免。一免后以及二免后的14 d分别通过颌下采血方法采血,通过间接ELISA方法检测各组小鼠血清中的特异性抗体,结果显示,二免后14 d,免疫组小鼠血清中CdtB、OPPA、Afua抗体水平分别与PBS+ISA201VG对照组和免疫之前比较均极显著提高(P<0.0001);利用PCV2免疫过氧化物酶单层试验(IPMA)抗体检测试剂盒检测各组小鼠血清抗体,结果显示在攻毒前PBS组均未检测到PCV2抗体,免疫组二免后14 d抗体水平为1∶800。二免后14 d,将免疫组和PBS组各随机分为3组(每组10只小鼠)进行攻毒保护试验,结果显示二联苗对PCV2攻毒组小鼠的免疫保护率为80%,对HPS5型HN10株攻毒组小鼠的免疫保护率为70%,与对照组比较差异极显著(P<0.01);对HPS13型ZD12株攻毒组小鼠的免疫保护率为80%,与对照组比较差异极显著(P<0.001)。上述结果首次表明,原核表达的rCap具有良好的免疫原性,PCV2-HPS二联亚单位疫苗对小鼠具有一定的保护效果,为后续PCV2-HPS二联亚单位疫苗的研究奠定了实验基础。

To evaluate the protective effect of porcine circovirus type 2-Haemophilus parasuis binary subunit vaccine(hereafter referred to as binary subunit vaccine)in mice,in this study,the PCV2 cap gene was cloned into the pMAL-c5X vector,and the recombinant Cap protein(rCap)was expressed by the prokaryotic system.The reactogenicity of the prokaryotic expressed Cap protein was demonstrated by western blot assay(using PCV2 monoclonal antibody as primary antibody).Using the recombinant proteins rCdtB,rAfua and rOPPA of Haemophilus parasuis(HPS),which had been constructed and preserved in our laboratory and emulsified with rCap protein and the adjuvant ISA201VG to make PCV2-HPS binany subunit vaccine.The concentration of each protein(rCdtB,rAfua,rOPPA and rCap)in PCV2-HPS binary subunit vaccine prepared in this study was 50μg/300μL.Sixty BALB/c mice at 6 weeks of age were randomly divided into two groups,the immunization group and the control group,and immunized by dorsal multi-point injection with a 14 days interval between the first and second immunizations.Blood was collected by submandibular blood collection after 14 days post the first immunization and the second immunization,and the serum of mice in each group was measured by indirect ELISA.The results showed that 14 days after the second vaccination,the antibody levels of CdtB,OPPA,and Afua in mouse sera in binary subunit vaccine immunization group were highly significant(P<0.0001)compared with the PBS+ISA201VG negative control group and those of before immunization,respectively.The PCV2 IPMA(Immunoperoxidase monolayer assay)antibody test kit was used to detect serum antibodies in various groups of mice.The results showed that the antibody level of rCap was 1∶800 in the binary subunit vaccine immunization group at 14 days after immu-nization.And before the virus challenge the antibodies were not detected in any of the PBS groups.Furthermore,after the second immunization for 14 days,the immunized and PBS groups were each randomly divided into three groups(10 mice in each group).The protection rate of the binary subunit vaccine was 80%against the challenge of PCV2 compared with no protection in the negative control group.The protection rate of binary subunit vaccine was 70%against the challenge of HPS serotype 5 HN10 strain,which was significantly different from that in the control group(P<0.01).Besides,the protection rate of binary subunit vaccine was 80%against the challenge of HPS serotype13 ZD12 strain,which was significantly different from that in the control group(P<0.001).The result shows that the prokaryotic expressed rCap protein has good immunogenicity and the PCV2-HPS binary subunit vaccine has good protective efficacy against the challenge with PCV2 and Haemophilus parasuis in mice model,which laid the experimental foundation for the subsequent study of PCV2-HPS binary subunit vaccine.