表达柔嫩艾美耳球虫HMGB1基因质粒DNA的免疫效果分析

Protective Immunity Induced by a Plasmid Encoding Eimeria tenella High Mobility Group box1 Gene against Homologous Challenge

旨在评价表达柔嫩艾美耳球虫(E.tenella)HMGB1基因质粒DNA免疫对同源株攻虫的免疫保护效果。将EtHMGB1基因插入pcDNA3.1(-)/myc-His A真核表达载体中,并在Hela细胞中进行体外瞬时表达。设计了pcDNA3.1(-)/myc-EtHMGB1免疫组、pcDNA3.1(-)/myc-His A免疫组、重组蛋白质+FCA佐剂免疫组、pcDNA3.1(-)/myc-EtHMGB1+重组蛋白质联合免疫组、FCA佐剂免疫组、未免疫攻虫组和未免疫未攻虫组7个试验组。分别于14和21日龄对鸡进行两次免疫,28日龄时除未免疫未攻虫组外各组用1×104个E.tenella孢子化卵囊攻虫,以平均增重、卵囊产量和病变记分作为免疫保护效果的评价指标。结果显示,成功构建了pcDNA3.1(-)/myc-EtHMGB1重组质粒,转染后该质粒可在Hela细胞中进行瞬时表达。该重组质粒单独免疫、重组蛋白质单独免疫或重组质粒与重组蛋白质联合免疫均对鸡肠道病变改善效果不明显,但可显著提高增重,降低卵囊产量,尤以重组质粒与重组蛋白质联合免疫效果最佳,显示Prime-boost免疫策略可提高该重组质粒的免疫保护效果。上述结果显示HMGB1可作为未来球虫疫苗研制的良好候选抗原之一。

This experiment was conducted to assess the immuno-efficacy of the eukaryotic plasmid.The high mobility group box1 gene fromEimeria tenella was inserted into expression vector pcDNA3.1（-）/myc-His A,and then constructed the eukaryotic expression recombinant plasmid pcDNA3.1（-）/myc-EtHMGB1 and expressed transiently the objective protein in Hela cells.Seven experimental groups including pcDNA3.1（-）/myc-EtHMGB1 immunization group,pcDNA3.1（-）/myc-His A immunization group,recombinant antigen with adjuvant immunization group,pcDNA3.1（-）/myc-EtHMGB1 with recombinant antigen prime-boost immunization group,FCA adjuvant immunization group,unimmunized and challenged group and unimmunized and unchallenged group were designed.Chickens were vaccinated at the age of 14 and 21days.All birds except the unchallenged control group were challenged orally with 1×10^4 E.tenellasporulated oocysts at 28 days.Gain of body weight,fecal oocyst output and lesion scores were assessed as measures of protective immunity.The results showed that the plasmid pcDNA3.1（-）/myc-EtHMGB1 was successfully constructed,and the transient expression product of EtHMGB1 could be detected by IFAand Western blot.Challenge experiments demonstrated that pcDNA3.1（-）/myc-EtHMGB1 immunization,recombinant antigen with adjuvant immunization and pcDNA3.1（-）/myc-EtHMGB1 with recombinant antigen prime-boost immunization could all increase body weight gains and reduce oocyst excretion,but all three methods did not have an effect on cecal lesion.The primeboost immunization strategy were superior to the other method based on the immune effects.These results suggest that EtHMGB1 could be proposed as an anti-apicomplexan vaccine candidate.