摘要
目的在构建基因工程菌pQE30-HpaA-DH5α的基础上,制备HpaA重组蛋白,以此为抗原,制备抗HpaA的蛋黄抗体(HpaA IgY)。通过小鼠口服试验,验证HpaA IgY阻止H.pylori在胃内定植的作用,为研制预防H.pylori感染的制剂奠定基础。方法大量诱导工程菌pQE30-HpaA-DH5α,获得重组蛋白HpaA,免疫产蛋立克体鸡,以水稀释法联合硫酸铵沉淀法提纯IgY,SDS-PAGE分析纯度,Bradford法测定浓度,Western blot鉴定对相应抗原的特异性,ELISA法检测效价。建立H.pylori感染Balb/c小鼠的动物模型,预防组在小鼠灌喂H.pylori菌液前灌喂不同剂量的HpaA IgY。组织学检查和细菌培养观察H.pylori定植。结果提纯后的HpaAIgY纯度为90%,浓度为24.6mg/mL,Western blotting证实有良好抗原结合特异性,ELISA效价为1∶12800。阳性对照组H.pylori的感染率为70.4%,12周后的感染率为88.9%。预防组的感染率明显低于阳性对照组,随IgY剂量的增加,感染率降低。IgY的剂量为6mg/mL时,能达到预防效果。结论成功制备出高纯度、高浓度、高效价的特异性HpaA IgY,在小鼠体内能阻止H.pylori的定植,有望进一步开发成为预防H.pylori感染的制剂。
Objective To prepare HpaA IgY against Helicobacter priori from yolk of egg laid from hen immunized with recombinant protein HpaA, based on constructed gene engineering bacteria pQE30-HpaA-DH5α of Helicobacterpylori. Methods After pQE30-HPaA-DH5α was cultured, mass HpaA recombinant proteins were induced and expressed, and then purified by Ni^2＋ -NAT chromatography. Laying hens were immunized with the fusion protein HpaA and the obtained IgY was isolated using method of the water dilution combined ammonium sulfate precipitation. The purity, titer, protein content, and specificity of IgY were detected by SDS-PAGE, ELISA, Bradford, and Western blotting, respectively. Before infection with H. pylori, Balb/c mice were administrated with different dosages of HpaA IgY by using an oral feeding needle in the prevention group. The planting of bacteria in stomach was assayed by observing bacterial culture and pathological section. Results HpaA recombinant proteins were expressed and IgY was prepared successfully. The concentration and purity of IgY were 24.6 mg/mL and 90%, respectively. The titer of IgY was 1：12 800. The total rate of infection in the positive control was 70.4% and after 12 weeks the infection rate was raised to 88.9%. The infection rate of the prevention group was lower than that of the positive control group at the same infected period, and was negatively related to the addition of IgY. The dosage of HpaA lgY that can prevent the infection of H. pylori was 6 mg/mL. Conclusion HpaA IgY with high concentration, high purity, high titer, and specificity is successfully prepared, which is able to inhibit the adhesive attraction of H. pylori in vivo and be used to prevent H. pylori infection.
出处
《免疫学杂志》
CAS
CSCD
北大核心
2008年第5期522-526,共5页
Immunological Journal
基金
重庆市教委渝教科(2003-7-3)
重庆市科委(CSTC
2005EA5020)基金资助
