G9P[8]型轮状病毒HN-28灭活病毒在小鼠中的免疫原性和保护效力评价

Evaluation of immunogenicity and protective efficacy of inactivated G9P[8] Rotavirus, HN-28 in mice

目的 将G9P[8]型轮状病毒HN-28灭活后与弗氏佐剂配伍免疫小鼠,评价其免疫原性和保护效力。方法 将冻融1次的HN-28病毒收获液离心去除细胞碎片,上清用50%蔗糖垫底进行超速离心,用MEM培养液重悬沉淀得到纯化的病毒,通过SDS-PAGE和Western blot分析纯化后的病毒;然后用甲醛灭活病毒,将灭活病毒在MA-104细胞上连续盲传代3次,通过观察细胞病变(cytopathic effect, CPE)和半数细胞感染病变法(CCID_(50))进行病毒灭活验证检测;将灭活病毒与弗氏佐剂按1∶1比例混合,免疫BALB/c小鼠,对照组用MEM与弗氏佐剂按1∶1比例混合后按相同方法和剂量免疫BALB/c小鼠。定期采血并用ELISA检测小鼠血清中抗轮状病毒IgG和IgA滴度;3剂免疫后用病毒微量中和试验测定每剂免疫后血清中和抗体并计算几何平均滴度(geometric average titer, GMT);3剂免疫后28 d,用HN-28活病毒灌胃攻毒,评价血清抗体对小鼠的保护效力。结果 SDS-PAGE和Western blot分析结果显示,纯化后的病毒纯度较高,在预期分子量处出现了对应的条带;HN-28灭活病毒在MA-104细胞连续传3代,显微镜下均未观察到CPE,CCID_(50)检测结果为阴性;免疫小鼠血清检测结果显示,HN-28灭活病毒在小鼠体内诱导产生的IgG抗体平均滴度为4.53 lg、IgA抗体平均滴度为2.54 lg、中和抗体平均滴度为2.86 lg,而对照组和免疫前血清中IgG、IgA均为阴性;与实验组小鼠相比,攻毒后1周,对照组小鼠体温明显升高,腹泻率增加,对照组小鼠粪便中轮状病毒的检出率高于实验组。结论 HN-28灭活病毒在BALB/c小鼠中具有良好的免疫原性和保护性。

Objective To evaluate the immunogenicity and protective efficacy of inactivated G9P [8] rotavirus HN-28 strain in BALB/c mice. Methods Virus culture was harvested after freeze-thawed once, and cell debris was removed by centrifugation. The concentrated virus was obtained by ultracentrifugation of the supernatant through a 50% sucrose cushion, and resuspend the precipitated pellet with MEM medium to obtain purified viruses. The purified virus was inactivated by formaldehyde, and virus inactivation was confirmed by infection of MA-104 cells with inactivated virus for three consecutive blind passages and subsequently assessed by CPE and CCID_(50). The inactivated virus was mixed with Freund's adjuvant in a ratio of 1∶1 to immunize BALB/c mice, and MEM was used as control. The titer of anti-rotavirus IgG and IgA in mouse serum were detected by enzyme-linked immunosorbent assay(ELISA). After 3 doses of immunization, the neutralizing antibodies in serum were detected by microneutralization assay and the geometric average titer(GMT) was calculated. On the 28th day after 3 doses of immunization, HN-28 live virus was administered by gavage to evaluate the protective effect of immune antibodies on mice. Results The results of SDS-PAGE and Western blot analysis showed that the virus was highly purified. The results of CPE and CCID_(50) confirmed that the virus was successfully inactivated. The average titers of IgG,IgA and neutralizing antibody induced by HN-28 inactivated virus in mice were 4.53 lg, 2.54 lg, and 2.86 lg, respectively. Compared with the experimental group, after 1 week of challenge, the body temperature and diarrhea rate of mice in the control group increased significantly, and the detection rate of rotavirus in feces of mice in the control group was higher than that of the experimental group. Conclusion The inactivated rotavirus HN-28 strain showed good immunogenicity and protective efficacy in BALB/c mice.