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目的评价不同方法纯化柯萨奇病毒A组16型(Coxsakievirus A 16,CA16)的可行性和有效性。方法将CA16病毒收获液经切向流超滤浓缩后,分别进行离心、氯仿抽提、分子筛和蔗糖垫层/梯度离心,按《中国药典》(三部)(2010版)的检定要求检测不同纯化方法得到的样品,检测指标包括:感染性滴度、抗原含量、蛋白含量、比活性、HPLC纯度、残余牛血清白蛋白、残余抗生素、免疫原性。结果从比活性来看,分子筛纯化法是其他方法的2~21倍,明显优于其他工艺;经HPLC检测,分子筛纯化样品纯度>98%,而其他工艺均在50%以下;分子筛法所得纯化样品中牛血清白蛋白残留量符合《中国药典》要求(<50ng/剂),去除率达99.97%;分子筛法和蔗糖垫层/梯度离心法所得纯化样品中抗生素残留量符合《中国药典》要求(<50ng/剂),分子筛法去除率在80%以上;从免疫小鼠后的抗体阳转率来看,离心和氯仿抽提样品组阳转率为100%,分子筛组为80%,蔗糖离心组为50%;从抗体几何平均滴度(geometric mean titer,GMT)来看,氯仿抽提组最高,为1∶912。结论以Sepharose 6Fast Flow作为层析介质的分子筛法可得到高纯度的CA16病毒,综合分析,该法是CA16较为理想的纯化方法
Objective To evaluate the feasibility and effectiveness of different methods for the purification of Coxsackievirus A 16 (CA16). Methods After the CA16 virus solution was concentrated by tangential flow ultrafiltration, the samples were centrifuged, extracted with chloroform, centrifuged with molecular sieve and sucrose cushion / gradient centrifugation, and tested according to the requirements of the Chinese Pharmacopoeia (2010) Samples obtained by the purification method, the detection indicators include: infectious titer, antigen content, protein content, specific activity, HPLC purity, residual bovine serum albumin, residual antibiotics, immunogenicity. Results From the specific activity, molecular sieve purification method is 2 ~ 21 times higher than other methods, which is obviously superior to other techniques. The purity of the sample purified by molecular sieve is more than 98% by HPLC, while the others are less than 50% The residue of bovine serum albumin in the sample meets the requirement of “Chinese Pharmacopoeia” (<50ng / dose) and the removal rate reaches 99.97%. The antibiotic residues in purified samples obtained by molecular sieve method and sucrose cushion / gradient centrifugation comply with the requirement of “Chinese Pharmacopoeia” (<50ng / dose), molecular sieve removal rate of more than 80%; immunization mice antibody positive rate of view, centrifugal and chloroform extraction group positive rate was 100%, molecular sieve 80%, sucrose The centrifugal group was 50%. From the geometric mean titer (GMT), the highest chloroform extract was 1: 912. Conclusion High purity CA16 virus can be obtained by using molecular sieve Sepharose 6 Fast Flow as chromatographic medium. The comprehensive analysis shows that CA16 is an ideal purification method