灭活SARS病毒的免疫原性的实验研究

An Experimental Study of Immunogenicity of Inactivated SARS Coronavirus in Mice

研究灭活SARS病毒的免疫原性。SARS病毒F6 9株经甲醛灭活后 ,加入氢氧化铝佐剂 ,以 3种剂量接种BALB/c小鼠 ,定时采血 ,测定特异性抗体的滴度及其中和活性 ,同时用化学发光酶联免疫法测定抗血清与SARS病毒结构蛋白的反应特异性及其相对强度。结果发现 ,小鼠接种疫苗 4d后 ,血清中可检测到IgM抗体 ,直至 2 6d后逐渐下降 ;IgG抗体在初次免疫 8d后出现 ,4 7d时达最高峰 ,6 3d后进入稳定期 ;不同剂量组的抗体滴度具有明显剂效关系 ,低剂量组和中剂量组滴度峰值为 1∶192 0 0 ,高剂量组滴度峰值为 1∶384 0 0。中和实验结果表明 ,小鼠所产生的抗体具有中和病毒活性 ,在 6 3d时 ,低剂量组和中剂量组血清的中和效价为 1∶12 80 ,高剂量组血清的中和效价为 1∶5 12 0。抗体分类结果表明 ,小鼠抗血清中含有针对多种SARS病毒结构蛋白的特异性抗体 ,其中 ,针对N蛋白、S4蛋白和S2蛋白的抗体水平相对较高 ,而抗M抗体、抗 3CL抗体的水平相对较低。上述结果说明 ,SARS病毒F6 9株经甲醛灭活后 ,各主要结构蛋白仍保持较强免疫原性 ;免疫小鼠后 ,可以诱导产生高滴度的特异性混合抗体 。

To study the immunogenicity of inactiv ated SARS coronavirus(SARS-Cov), F69 strain was treated with formaldehyde and mixed with Al(OH) 3 so as to the inactivated SARS-Cov vaccine. Three groups of pathogen-f ree BALB/c mice were challenged using the vaccine at three times(da y 0, day 14, and day 24). The vaccine contained either 2.5×10 5, 5×105, or 7.5×105 TCID 50 of SARS-Cov. Sera of mice were col lected from the tail vein after day 0 of immunization, and the titer of IgM and IgG against SARS-Cov were measured, the neutralization activity was determined, as well as the specificity and the relative binding strength of antibodies to SARS-Cov proteins were measured by CLEIA method on a protein chip. It was found the mice exhibited IgM antibody on day 0 and the level of IgM antibody did not declined until day 26. The IgG antibody appeared of mice sera on day 8, peaked on day 47 and went into plat form phase on day 63. The levels of IgG antibody in different mice groups exhibited significant dependence relation to the injection dose. The peak titer of IgG in low-dose group and middle-dose group was 1∶19 200, in high-dose group it was 1∶38 400. Neutralization assay demonstrated that the neutralization titer on day 63 of low-dose group and middle-dose group was 1∶1 280, of high-dose group it was 1∶5 120. Protein chip test indicated that the mice antisera was a mixture of antibodies specific to different proteins of SARS-Cov, among which the antibodies specific to N protein and 3CL protein was lower than others. Those r esults suggest that the inactivated SARS-CoV preserve its antigenicity and vaccine prepared with this inactivated virus induces mice to produce high level of antibodies, which might protect host cell from SARS-Cov's challenge.