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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 botulinum toxin type E by different techniques and to detoxify the toxoid and to analyze its immunogenicity. Methods The botulinum toxin type E was prepared by intracellular extraction method and acid precipitation method respectively. After toxin type specific assay and virulence determination, the detoxification method was used to prepare toxoid. According to the Chinese Pharmacopoeia (2010 edition) , Protein nitrogen (PN) content, total nitrogen (TN) content, p H determination and detoxification test and toxicity reversal test. Three groups of toxins were selected to prepare three groups of antigens. The rabbits were given basic subcutaneous immunization (2-needle) and 3-way hyperimmune (2-needle / d, were all on the 20th day after the second immunization, blood was collected via the ear vein and the serum was separated. The antibody titers were determined by reference to the titration of the botulinum toxin antitoxin in Appendix III of the Chinese Pharmacopoeia (2010 edition). Results The toxin (201112001 and 201112003) and 55,120 h acid precipitated toxin (batch of 201112002 and 201112004) were all botulinum toxin type E in 55 and 120 h, and their virulence was 6.2 × 103 and 5.0 × 102, respectively. 4.5 × 102,6.5 × 103 LD50 / ml. The detoxification test and toxicity reversal test results of the toxoid in each group were in accordance with the provisions of the Chinese Pharmacopoeia (2010 edition), and the contents of TN and PN The highest, 120 h bacteria to extract the lowest toxin. The antigen binding costs of the three groups of antigens (201112001-1, 201112004-1 and 201112004-2) prepared were 900, 2100 and 900 BU / ml, respectively. The toxoid prepared by the two methods was formulated according to the highest binding value, and after immunization (P> 0.05). The toxoid prepared by the two methods had the same binding value and different binding antigen with the same batch of toxoid, and the differences in the titer of serum after immunization were statistically significant Significance (P <0.05). Conclusion Acid-precipitation method was used to extract toxins from the 120 h culture medium to obtain the toxoid with the same immunogenicity as the 55 h bacteria extract. Acid precipitation method is simple, more suitable for the immunity of horses with E-type meat antigen production scale.