摘 要

脊髓灰质炎（简称脊灰）是由脊髓灰质炎病毒引起，主要通过粪-口途径传播的急性传染病，俗称小儿麻痹症。疫苗是预防该疾病的有效手段。血清支持细胞的生长和繁殖能力是细胞培养过程中评价血清质量最主要的指标之一，它的选择对细胞和病毒的培养具有十分重要的意义。疫苗的免疫效果与病毒含量密切相关，所以准确测定其含量是疫苗有效性的保证。目前，使用血清试剂对脊髓灰质炎病毒培养及检测结果可能存在的影响是不容忽视的。且市面上不同来源不同厂家的血清在质量上存在批间差异，增加了疫苗生产和检定的不稳定性和疫苗质量控制的难度。

目的：研究血清对脊髓灰质病毒培养及脊髓灰质炎病毒感染性滴度检测的影响。

方法：（1）用不同血清适应Vero细胞培养后，以三种M.O.I接种脊髓灰质炎病毒并收获病毒，采用微量滴定法测定病毒收获液感染性滴度，采用ELISA法检测收获液D抗原，分析不同血清培养对脊髓灰质炎病毒培养的影响。（2）采用6种未处理血清和6种预处理血清组（免疫吸附法）进行脊髓灰质炎病毒感染性滴度检测，检测了不同组血清中脊髓灰质炎病毒中和抗体效价，并使用处理血清组进行促细胞生长试验，分析不同血清对感染性滴度的影响。

结果：（1）不同血清培养Vero细胞，细胞形态相似，细胞计数结果差异不显著（P＞0.05），倍增时间差异不明显（P＞0.05），病毒滴度差异不显著（P＞0.05），D抗原检测结果，I型、III型进口胎牛血清优于进口新生牛血清、进口小牛血清和国产新生牛血清，差异显著（P＜0.05）；II型差异不显著（P＞0.05）。相同血清不同M.O.I接种脊髓灰质病毒，病毒滴度和D抗原含量无影响，只是影响病毒病变时间快慢。（2）6种未处理血清组病毒感染性滴度检测结果差异显著（P＜0.01），6种处理血清组病毒感染性滴度差异不显著（P＞0.05）。未处理组2号血清的中和抗体效价为1:4，6号血清的中和抗体效价为1:8，其余各组血清的中和抗体效价均＜1:4。6种处理血清组的中和抗体效价均为1:2。血清促细胞生长试验显示6种处理组血清细胞生长趋势正常。

结论：（1）不同血清培养细胞会影响之后培养病毒的质量和产量，提示可优化培养条件，改进增加细胞密度的可能，更有效的利用生物反应器生产SabinIPV。（2）比较6种不同血清对脊髓灰质炎病毒感染性滴度（CCID₅₀）检测结果的影响。脊髓灰质炎病毒感染性滴度检测前使用免疫吸附对血清进行预处理，可降低血清中存在的中和抗体对检测的影响，为比较不同实验室数据提供可能。

ABSTRACT

Poliomyelitis(Polio) is an acute contagious fecal-oral transmitted disease caused by polio virus, commonly known as infantile paralysis. Vaccine is the only effective method for prevention. The ability supporting cells growth and reproduction in the process of cell culture of serum is one of the primary indicator of its quality. Choosing a kind of high quality serum using in cells and virus culture is significant. The immunogenicity of vaccines dues to its virus contents thus, precise measurement of the virus contented in the vaccine ensures the immune effects of vaccines. At the present, the possible effects of using serum reagents for polio virus culturing and testing cannot be ignored. There have being some difference between different batch of serum selling on the market, which has increased the instability of vaccine production and verification and has increased the difficulty of vaccine quality control.

  Objective: Study serum influence on poliovirus culture and poliovirus titer test.

Method:(1) Each serum adapt to Vero cell, poliovirus was inoculated in Vero cells with 3 different M.O.I and harvested, the influence of different serum to poliovirus culture was analyzed by measuring infectious titer of virus harvest fluid by micro measurement method and D antigen by ELISA method. (2) By using 6 kinds of untreated serum and 6 kinds of pretreatment serum group respectively, the degree of polio virus infectious droplets had been tested. Also, the poliovirus neutralization antibody titer of different group had been tested. After that, we presented the cell growth experiment in the pretreatment serum group. We analyzed the influence on CCID₅₀(cell culture infective dose 50%) data from different chosen serum.

Results:(1)Different serum culturing Vero cell, cell morphology, cell calculation result difference was not eminent(P＞0.05), doubling time has no significant difference(P＞0.05). Virus titer has no significant difference (P＞0.05). D antigen test shows type one and type three imported fetal calf serum has significant difference with imported new-born calf serum, imported calf serum and domestic new-born calf serum the rest have significant difference (P＜0.05),type two the rest have no significant difference (P＞0.05). When inoculating poliovirus with same serum but different M.O.I, virus titer and D antigen content was not influenced and virus lesion time was influenced.（2）According to the final results, the difference of polio virus CCID50between these 6 kind of untreated serum groups were significant（P<0.01）, however, the contrary results could be seen in the pretreatment serum groups, which had an significant difference(P＞0.05).In these untreated serum groups, NO.2 serum sample had a 1:4 neutralizing antibody titer, the NO.6 serum sample had a 1:8 neutralizing antibody titer, and the others had a neutralizing antibody titer less than 1:4.The neutralizing antibody titer all the 6 kinds of pretreatment serum were 1:2. In the cell growth experiments, 6 kinds of pretreatment serum could maintain the cell normal growth.

Conclusions:(1) In analysis on different calf serum influence Vero cell growth in Vero cell culture leads to influence virus production and quality, we can optimize culture condition in a better way. By optimizing cell density augment and may lead to more effective capacity of Sabin IPV production in bioreactor. (2) Influence on poliovirus CCID50 (cell culture infective dose 50%) by different serum has been compared. Using the serum immune adsorption method to pretreat the serum before Polio virus infectious droplets degree testing can reduce the influence of neutralizing antibody in the serum, which could be a possibility to comparing different experiment data between different laboratory.