CN103948622B - 羧甲基葡聚糖在制备提高禽流感疫苗抗体阳性率药物中的应用 - Google Patents
羧甲基葡聚糖在制备提高禽流感疫苗抗体阳性率药物中的应用 Download PDFInfo
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Abstract
本发明公开了羧甲基葡聚糖(CMG)在制备提高禽流感疫苗抗体阳性率药物中的应用。所述的应用是将羧甲基葡聚糖与矿物油乳化成油乳剂剂型后作为佐剂预先或注射疫苗时注入动物机体内。本发明可以显著提高禽流感疫苗抗体阳性率,从原来的65%左右提高到100%,且维持时间长,有效降低感染发病的几率。其次,本发明无任何毒副作用、无残留、使用方便,易于控制用量。
Description
技术领域
本发明涉羧甲基葡聚糖,具体来说涉及羧甲基葡聚糖在制备提高禽流感疫苗抗体阳性率药物中的应用。
背景技术
目前,灭活疫苗在畜禽疫病防控中发挥重要作用,但现有灭活疫苗免疫机体后,普遍存在抗体阳性率较低,抗体持续期较短等问题。一般常规灭活疫苗需要免疫两次以上,且免疫后2-3周才能对机体提供有效保护。在规模化养殖条件下,因畜禽免疫后抗体阳性率较低,无法提供有效保护,可能会波及整群发病,带来严重经济损失。
禽流感是危害我国养禽业发展重要传染病之一,免疫灭活疫苗是防控这种疾病有效措施之一。目前,由于鸡群免疫抑制疾病发病率较高,禽流感疫苗免疫后抗体阳性率普遍较低,特别是在肉鸡上体现尤为明显。某些肉鸡饲养场,H5亚型禽流感抗体阳性率为0,存在严重的免疫缺陷。另外,肉鸡生长期比蛋鸡短,且免疫性能方面比蛋鸡略差,在母源抗体消失至免疫后自身产生有效保护抗体需要一段时间,同时肉鸡生产期只有40多天,对疫苗的免疫效力要求更高。因此,临床生产上亟需一种能提高现有禽流感灭活疫苗抗体效价的药物,以提高现有灭活疫苗对肉鸡的保护。
羧甲基葡聚糖是酵母葡聚糖的羧甲基化衍生物,是一种新型高效、无毒、水溶性生物大分子,它具有广泛的生理调节作用。例如1)促进伤口愈合:研究发现使用羧甲基葡聚糖涂抹动物腿部伤口,能明显加速其伤口愈合。2)晒后修复:研究发现羧甲基葡聚糖能够快速修复因紫外照射导致的皮肤红斑。3)抗衰老:皮肤中具有免疫活性的朗格罕氏细胞能够被羧甲基葡聚糖活化,诱发其系统的免疫和修复功能。羧甲基葡聚糖已被列入国际化妆品原料名单(INCI),广泛应用化妆品领域,然而,尚未有关于羧甲基葡聚糖提高禽流感疫苗抗体阳性率的报道。
发明内容
本发明公开了羧甲基葡聚糖在制备提高禽流感疫苗抗体阳性率药物中的应用。它是将羧甲基葡聚糖与矿物油乳化成油乳剂后,按照一定比例与商品疫苗混合后注入畜禽机体内。其中所述的提高禽流感疫苗抗体阳性率指的是提高肉鸡H5亚型禽流感疫苗抗体的阳性率。
本发明所述的羧甲基葡聚糖指的是含有羧甲基葡聚糖的油乳剂型免疫增强剂。特别是应用于家禽时,在制备的油乳剂型免疫增强剂中羧甲基葡聚糖最终浓度为5g/100mL。
本发明所述的羧甲基葡聚糖,指的是含有羧甲基葡聚糖的油乳剂型免疫增强剂。
本发明进一步公开了油乳剂型免疫增强剂,它是由下述重量份数的原料组成:
羧甲基葡聚糖10g吐温-802mL
白油94mL司班-806mL
注射用水100mL。
本发明同时也公开了油乳剂型免疫增强剂的制备方法按如下的步骤进行:
(1)配制水相溶液:将10g羧甲基葡聚糖加入100mL注射用水中溶解,然后加入吐温-802mL,200rpm搅拌10min,配制成水相溶液。
(2)配制油相溶液:白油与司班-80混合配制成油相溶液;其中白油:司班-80的体积比比为100:6。
(3)将配制的水相溶液缓慢加到油相溶液中,然后在24000r/min乳化10min,即得羧甲基葡聚糖油乳剂;其中水相溶液与油相溶液的体积比1:1。
本发明更进一步公开了油乳剂型免疫增强剂使用方法,它是将油乳剂型免疫增强剂与商品禽流感灭活疫苗按体积比1∶9混合后注入动物机体。该羧甲基葡聚糖油乳剂能显著提高H5亚型禽流感商品疫苗抗体抗体阳性率,且高达100%。
本发明更加详细的试验方法及结果如下:
1材料和方法
1.1疫苗和检测试剂盒
H5(Re-5)亚型禽流感商品疫苗,购自哈尔滨维科生物技术开发公司。禽流感抗体ELISA检测试剂盒,购自美国爱德士(IDEXX)试剂公司。
1.2试验动物
1日龄肉鸡购自天津静海县某肉鸡养殖场,在普通环境中饲养。
1.3油乳剂免疫增强剂配制及使用方法
(1)配制水相溶液:在注射用水中分别加入不同克数羧甲基葡聚糖,然后加入2%吐温-80,200rpm搅拌,10min,配制成水相溶液,使其终浓度分别为;50g/100mL,5g/100mL,0.5g/100mL。
(2)配制油相溶液:94%白油与6%司班-80混合配制成油相溶液。
(3)将水相以1∶1的比例缓慢加到油相中后,24000r/min乳化10min,即得油乳剂型免疫增强剂。
(4)将油乳剂型免疫增强剂与商品禽流感灭活疫苗按体积比为1∶9混合,注入动物机体进行免疫。
1.4动物免疫
选择7日龄肉鸡禽流感母源抗体效价为0鸡群。将高中低三种浓度油乳剂免疫增强剂按照1:9与H5(Re-5)亚型禽流感商品疫苗混合后,共同免疫(分别标记为G、Z、D组),免疫鸡日龄为7日龄。另设常规疫苗(记为MD组)对照,疫苗剂量为0.3mL/只鸡,20只鸡/组。免疫后第1周、第2周、第3周、第4周和第5周采血,抗体检测。采用ELISA试剂盒检测抗体阳性率。
2结果与讨论
2.1肉鸡免疫后的抗体阳性率
从图1,2中可以看出,在免疫后第2周,所有试验组H5亚型禽流感抗体阳性率均达70%,各组之间均无明显差异;在免疫后第3周,SPF雏鸡(Z组)抗体阳性率达100%,且一直持续到免疫后5周(42日龄),而常规疫苗免疫对照组(MD组)与低剂量组(D组)抗体阳性率为80%,且在免疫后第4周抗体阳性率下降到62%左右;高剂量组(G组)在免疫后第2周抗体阳性率达70%,且一直持续到免疫后第5周。
试验结果表明,中剂量(5g/100mL)羧甲基葡聚糖油乳剂能显著提高H5亚型禽流感疫苗抗体抗体阳性率,且高到100%,与常规疫苗和其他剂量羧甲基葡聚糖油乳剂相比较,能维持到免疫后第5周(42日龄)。由此可见,从长效的预防机制上面来看,本发明可以提高H5亚型禽流感疫苗的抗体阳性率达100%,且持续到42日龄,从而在肉鸡饲养全过程中,有效地预防禽流感疾病发生。
附图说明:
图1各试验组鸡免疫后不同时间抗体S/N值平均值;
图2各试验组鸡免疫后不同时间抗体阳性率。
具体实施方式
下面结合具体实施例,进一步阐述本发明。应理解这些实施例仅用于说明本发明而不用于限制本发明的范围。
实施例1
50g/100mL羧甲基葡聚糖油乳剂免疫增强剂配制
(1)配制水相溶液:将100g羧甲基葡聚糖加入100mL注射用水中溶解,然后加入吐温-802mL,200rpm搅拌10min,配制成水相溶液。
(2)配制油相溶液:白油与司班-80混合配制成油相溶液;其中白油:司班-80的体积比比为100:6。
(3)将配制的水相溶液缓慢加到油相溶液中,然后在24000r/min乳化10min,即得羧甲基葡聚糖油乳剂;其中水相溶液与油相溶液的体积比1:1。
实施例2
5g/100mL羧甲基葡聚糖油乳剂免疫增强剂配制
(1)配制水相溶液:将10g羧甲基葡聚糖加入100mL注射用水中溶解,然后加入吐温-802mL,200rpm搅拌10min,配制成水相溶液,使其终浓度分别为5g/100mL。
(2)配制油相溶液:白油与司班-80混合配制成油相溶液;其中白油:司班-80的体积比比为100:6。
(3)将配制的水相溶液缓慢加到油相溶液中,然后在24000r/min乳化10min,即得羧甲基葡聚糖油乳剂;其中水相溶液与油相溶液的体积比1:1。
实施例3
对比试验
将5mg/100mL油乳剂免疫增强剂按照1:9与H5(Re-5)亚型禽流感商品疫苗混合后,共同免疫(发明组),免疫鸡日龄为7日龄。另设常规疫苗对照组(记为对照组),疫苗剂量为0.3mL/只鸡,20只鸡/组。免疫后不同时间段采血抗体检测。采用ELISA试剂盒检测抗体阳性率。
。
结论:
(1)相比对照组,本发明可以提高H5亚型禽流感疫苗的抗体阳性率达100%。
(2)相比对照组,本发明羧甲基葡聚糖油乳剂能提高H5亚型禽流感免疫持续期,且持续到42日龄,从而在肉鸡饲养全过程中,有效地预防禽流感疾病发生。
Claims (2)
1.羧甲基葡聚糖在制备提高禽流感疫苗抗体阳性率药物中的应用,其中所述的提高禽流感疫苗抗体阳性率指的是提高肉鸡H5亚型禽流感疫苗抗体的阳性率;所述的羧甲基葡聚糖指的是含有羧甲基葡聚糖的油乳剂型免疫增强剂,羧甲基葡聚糖最终浓度为5g/100mL。
2.权利要求1所述的应用,其特征在于它是由权利要求1所述的油乳剂型免疫增强剂与商品禽流感灭活疫苗按体积比1∶9混合使用。
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羧甲基葡聚糖注射剂药效学研究;孙英峰 等;《中国畜牧兽医》;20140120;第41卷(第1期);133-136 * |
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