不同工艺制备的E型肉毒毒素及其类毒素的免疫原性分析

Immunogenicity of Clostridium botulinum type E toxin and toxoid prepared by various processes

目的用不同工艺制备E型肉毒毒素,脱毒制备类毒素,并分析其免疫原性。方法分别用菌体内提取法和酸沉淀法制备E型肉毒毒素,进行毒素型特异性检定及毒力测定后,脱毒制备类毒素,参考《中国药典》三部(2010版)进行结合价、蛋白氮(PN)含量、总氮(TN)含量、p H测定及脱毒检查和毒性逆转试验。选择结合价较高的2批类毒素配制3组抗原,经家兔皮下进行基础免疫(共2针)和3程超免疫(2针/程),每针间隔14 d,每程免疫间隔30 d,均于第2针免疫后第20天,经耳缘静脉采血,分离血清,参考《中国药典》三部(2010版)附录ⅪH肉毒抗毒素效价检测法,检测抗体效价。结果 55、120 h菌体内提取毒素(201112001和201112003批)及55、120 h酸沉淀提取毒素(201112002和201112004批)均为E型肉毒毒素,毒力分别为6.2×103、5.0×102、4.5×102、6.5×103 LD50/ml;各组类毒素脱毒检查和毒性逆转试验结果均符合《中国药典》三部(2010版)规定,55 h酸沉淀提取毒素其类毒素TN、PN含量最高,120 h菌体内提取毒素最低。制备的3组抗原(201112001-1、201112004-1及201112004-2批)抗原结合价分别为900、2 100和900 BU/ml;两种方法制备的类毒素按最高结合价配制抗原,免疫后的血清效价差异无统计学意义(P>0.05),两种方法制备的类毒素按相同结合价配制抗原及同批类毒素配制的不同结合价抗原,免疫后的血清效价差异均有统计学意义(P<0.05)。结论用酸沉淀法从120 h培养液中提取毒素可获得与55 h菌体内提取毒素相同免疫原性的类毒素。酸沉淀法操作简便,更适宜马匹免疫用E型肉毒抗原的规模化生产。

Objective To prepare Clostridium botulinum type E toxin by various processes and further prepared toxoid by detoxication, then analyze the immunogenicity of prepared toxin and toxoid. Methods The toxin of Clostridium botulinum type E was prepared by intracellular extraction and acid precipitation respectively, identified for type specificity,determined for virulence and detoxified. The obtained toxoid was tested for valence,protein nitrogen（PN）content,total nitrogen（TN） content,p H value,detoxification efficacy and virulence reversion. Two batches of toxoid with high valences were prepared into three groups of antigen. Rabbits were immunized s.c. with two doses of the toxoid at an interval of 14 d for primary immunization, then boosted by three courses each consisting of two doses for hyperimmunization. The interval of courses was 30 d. Serum samples were collected on day 20 after the second dose,and determined for antibody titer by the method in Appendix Ⅺ of Chinese Pharmacopeia（Volume Ⅲ,2010 edition）.Results The toxins of Lots 201112001 and 201112003 prepared by intracellular extraction 55 and 120 h after culture and those by acid precipitation 55 and 120 h after culture（Lots 201112002 and 201112004） were identified as Clostridium botulinum type E,of which the virulence were 6. 2 × 10^3,5. 0 × 10^2,4. 5 × 10^2 and 6. 5 × 10^3 LD50/ ml respectively.The results of detoxification and virulence reversion tests of toxoids in various groups met the requirements in Chinese Pharmacopeia（Volume Ⅲ,2010 edition）. The TN and PN contents in toxoid prepared with toxin obtained by acid precipitation 55 h after culture were the highest,while those by intracellular extraction 120 h after culture were the lowest. The valences of three groups of prepared antigen（Lots 201112001-1,201112004-1 and 201112004-2） were900,2 100 and 900 BU / ml respectively. The serum antibody titers induced by antigens formulated with toxoids prepared by two methods,at the highest valence,showed no significant difference（P 〉0. 05）. However,the serum antibody titers induced by antigens formulated with toxoids prepared by two methods,at the same valences,as well as those by the antigens formulated with toxoids of the same batch,at various valences,showed significant difference（P 〈0. 05）. Conclusion The immunogenicity of toxoid prepared from the toxin obtained by acid precipitation after culture for 120 h was comparable to that by intracellular extraction 55 h after culture. However,acid precipitation was simple to handle and more suitable for large-scale production of Clostridium botulinum type E antigen for immunization in horses.