SS-HPS-APP三联亚单位疫苗对小鼠保护效果的评价

Evaluation of the protective efficacy of Streptococcus suis-Haemophilus parasuis-Actinobacillus pleuropneumoniae triple subunit vaccine on mice

为评价猪链球菌病-副猪嗜血杆菌病-猪传染性胸膜肺炎三联亚单位疫苗(下称三联苗)对小鼠的保护效果,本研究将猪链球菌(SS)保护性抗原EF和猪胸膜肺炎放线杆菌(APP)保护性抗原OMP、ApxI、ApxII序列分别克隆至pET-28a和pCold-sumo载体,再转化至E.coli BL21(DE3)中构建重组菌,经过诱导表达纯化获得重组蛋白r EF、rOMP、rApxI和rApxII。利用本研究室前期构建的pET-28a-sly/BL21、pET-28a-mrp/BL21、pET-28aoppa/BL21、pET-28a-oppa2/BL21、pET-28a-afua/BL21、pET-22b-cdtb/BL216株重组表达菌株,表达纯化SS的rSLY、rMRP和副猪嗜血杆菌(HPS)的r OPPA、rOPPA2、rAfuA、rCdtB。将上述重组蛋白等量混匀,使每一剂疫苗中各蛋白的含量均为20μg,再与ISA 201佐剂乳化制成三联苗。将血清2型SS(SS2)464株和SS9 GZ2株按照不同比例稀释后感染C57小鼠;将血清5型HPS(HPS5)HN10株、HPS13 ZD12株、血清5型APP(APP5)MD株和APP7 S-8株按照不同比例稀释后感染BALB/c小鼠,统计上述6个菌株对小鼠的最小致死剂量(MLD)。将20只C57小鼠和40只BALB/c小鼠均分成2组(每组含10只C57小鼠和20只BALB/c小鼠),间隔14 d分别皮下免疫2次300μL PBS+ISA 201佐剂和三联苗。二免后14 d采集小鼠血清,通过间接ELISA检测各组小鼠的EF、MRP、SLY、OPPA、OPPA2、CdtB、AfuA、OMP、ApxI和ApxII的抗体水平。首免后28 d,将C57小鼠再按照免疫组别平均分为2组,分别以MLD的SS2和SS9攻菌;同理,将BALB/c小鼠再平均分为4组,分别以MLD的HPS5、HPS13、APP5和APP7攻菌。结果显示:SS2和SS9对C57小鼠的MLD均为5×10^(7)cfu/只,HPS5、HPS13、APP5和APP7对BALB/c小鼠的MLD分别为1.5×10^(8)cfu/只、5×10^(7)cfu/只、1.5×10^(8)cfu/只和7.5×10^(8)cfu/只。与免疫前相比,二免后14 d实验组小鼠体内对EF、SLY、MRP、OPPA、OPPA2、CdtB、AfuA、OMP、ApxI和ApxII的抗体水平显著升高(p<0.0001),对照组小鼠体内对10种抗原的抗体水平均无差异;实验组与对照组二免后抗体水平差异显著(p<0.0001)。攻菌实验中,对照组小鼠在观察期内全部死亡,攻菌SS2、SS9、HPS5、HPS13、APP5和APP7的实验组小鼠存活率分别为100%(5/5)、80%(4/5)、60%(3/5)、80%(4/5),80%(4/5),100%(5/5),对照组和实验组的存活率有显著差异(p<0.05)。以上结果表明该三联苗对SS2、SS9、HPS5、HPS13、APP5和APP7攻击的小鼠能够提供良好的交叉保护性。本研究首次系统研究并证实了该三联苗的免疫保护结果较好,为其进一步研究及应用提供了实验依据。

To evaluate the protective effect of Streptococcus suis(SS)-Haemophilus parasuis(HPS)-Actinobacillus pleuropneumoniae(APP)triple subunit vaccine to mice,plasmids of pET-28 a-ef,pET-28 a-omp,p Cold-sumo-apxI and p Cold-sumo-apxII were constructed and transferred into E.coli BL21,then the recombinant proteins of rEF of SS,rOMP,rApxI and rApxII of APP were obtained by induction and purification.Six recombinant strains constructed in our laboratory pET-28 a-sly/BL21,p ET-28 a-mrp/BL21,pET-28 a-oppa/BL21,p ET-28 a-oppa2/BL21,pET-28 a-afua/BL21 and pET-22 b-cdtb/BL21)were used to express and purify rSLY and rMRP of SS,and rOPPA,rOPPA2,r AfuA and rCdtB of HPS in BL21.The above 10 proteins(20μg per protein in one dosage)were equally mixed and emulsified with ISA 201 VG to produce triple vaccine.After C57 mice were infected by 464 strain of serotype 2 SS(SS2)and GZ2 strain of serotype 9 SS(SS9)at different dilutions respectively,BALB/c mice were infected by HN10 strain of serotype 5 HPS(HPS5),ZD12 strain of serotype 13 HPS(HPS13),MD strain of serotype 5 APP(APP5),and S-8 strain of serotype 7 APP(APP7)at different dilutions respectively,the minimum lethal dose(MLD)for 6 strains to mice was calculated.20 C57 mice and 40 BALB/c mice were divided into two groups(10 C57 mice and 20 BALB/c mice in each group)and were immunized with 300μL PBS+ISA 201 VG and triple vaccine twice every 14 days respectively.The serum of mice was collected 14 days after the secondary immunization,and the antibody levels of EF,MRP,SLY,OPPA,OPPA2,CdtB,AfuA,OMP,ApxI,and ApxII in each group were detected by indirect ELISA.28 days after the first immunization,C57 mice were divided into2 groups from different immune groups,and challenged with MLD of SS2 and SS9 respectively;similarly,BALB/c mice were divided into 4 groups,and challenged with MLD of HPS5,HPS13,APP5 and APP7 respectively.The results showed that:the MLD of SS2 and SS9 on C57 mice was 5×10^(7)cfu/mouse,and MLD of HPS5,HPS13,APP5 and APP7 on BALB/c mice was 1.5×10^(8)cfu/mouse,5×10^(7)cfu/mouse,1.5×10^(8)cfu/mouse and 7.5×10^(8)cfu/mouse,respectively.After the second immunization,the antibody levels against EF,SLY,MRP,OPPA,OPPA2,CdtB,AfuA,OMP,ApxI,and ApxII in the experimental group were significantly increased(p<0.0001),while the control group didn’t changed.And the antibody levels between the experimental and control groups were significantly different(p<0.0001).In the challenge experiment,mice in control groups all died within the observation period.The survival rates of experimental groups to SS2,SS9,HPS5,HPS13,APP5,and APP7’s challenge were 100%(5/5),80%(4/5),60%(3/5),80%(4/5),80%(4/5),and 100%(5/5)respectively,which were significantly different from the control group(p<0.05).All the results indicated that the triple vaccine exhibited good cross protection against SS2,SS9,HPS5,HPS13,APP5 and APP7.This study is the first systematic research to verify the protective effect of the triple vaccine,which provides an experimental basis for further research and application.