抗铜绿假单胞菌IgY的制备优化及抑菌效果  

The optimized preparation of IgY against p.aeruginosa and the detection of its antibiotic effect

作　　者：姚羽菲 龙敏[2] 文荟淋 姚刚 

机构地区：[1]四川大学华西临床医学院,成都610041 [2]四川大学生命科学学院,成都610065 [3]成都安蒂康生物科技有限公司,610041

出　　处：《检验医学与临床》2018年第2期202-205,208,共5页Laboratory Medicine and Clinic

摘　　要：目的探讨高效价、高纯度且生物活性好的抗铜绿假单胞菌(PA)的特异性鸡卵黄免疫球蛋白(IgY)的制备工艺。方法采用超声破碎、共振裂解、研磨破碎等方法裂解PA,筛选出最佳裂解抗原,然后将裂解抗原或经甲醛灭活的全菌抗原与佐剂充分乳化免疫产蛋母鸡;采用酶联免疫吸附试验检测水稀释后的IgY抗体效价变化;通过硫酸铵盐析,超滤法纯化抗体,并用十二烷基磺酸钠聚丙烯酰胺凝电泳法(SDS-PAGE)和二喹啉甲酸(BCA)法测定抗体纯度和浓度,平板法评价IgY对PA的抑菌效果,并以蒸馏水作为对照;扫描电镜法检测细菌形态变化。结果超声破碎法裂解的PA抗原浓度最高且蛋白条带最为完整;0.5mL的1×109CFU/mL灭活菌初次免疫4周后再次免疫的方案产生的抗体效价最高,可高达1∶25 600;硫酸铵沉淀,超滤纯化后的抗体纯度可达94%,浓度为27.02mg/mL;IgY体外抑菌试验表明,2.7mg的抗体能明显抑制PA生长,与蒸馏水对照组相比,IgY试验组菌落数减少了9/10,并且明显抑制了细菌绿色色素的产生;扫描电镜观察IgY试验组菌体较长、较大且皱缩。结论采用灭活全菌免疫母鸡,经水稀释、硫酸铵沉淀、超滤纯化的制备方法可得到高纯度和高效价的抗PA IgY,所制抗体具有特异性和显著抑菌活性。Objective To prepare the pure and effective immunoglobulin yolk （IgY） against pseudomonas aeruginosa, and optimize the preparation process. Methods Using these methods, such as ultrasonication, res onance cracking, grinding, we lysed p. aeruginosa and gained the optimized lysis antigen. The laying hens were immunized with lysis antigen or inactivated bacteria. Indirect Enzyme-linked immunosorbent was used to eval uate the titer and stability of IgY. Then IgY was isolated and purified from egg yolks with high-titer using wa- ter dilution-salting out method or ultrafiltration. The purity and concentration of IgY were analysed by SDS- PAGE and BCA, respectively. The antibacterial effect was evaluated by plate method,and the cell morphology was detected by scanning electron microscopy. Results Ultrasonieation was the most effective way to crack the antigen of p. aeruginosa. Immunization scheme that 0.5 mL inactivate bacteria （15〈 109 CFU/mL） were in jeeted hens and secondly injected after 4 weeks was proved the best for the titer of 1 ： 25 600. The purity of IgY was 94% after ammonium sulfate precipitation and ultrafiltration purification and the concentration 27.02 mg/mL,2.7 mg IgY could restrain the growth of bacteria obviously and the colony number decreased 9/10 compared to the control group. Meanwhile, the green pigment produced by p. aeruginosa was inhibited obviously. Scanning electron microscope showed p. aeruginosa were bigger and shrinking after treated with IgY. Conclusion The preparation and purification IgY against P. aeruginosa has been successfully optimized. And the IgY antibody has specific and marked antibacterial activity.

关 键 词：铜绿假单胞菌 鸡卵黄抗体 抑菌效应 

分 类 号：Q816[生物学—生物工程]