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目的探讨人巨细胞病毒(human cytomegalovirus,HCMV)糖蛋白B(glycoprotein B,g B)中AD2位点Ⅰ(AD-2S1)在小鼠中的免疫效果。方法将AD-2和4×AD-2S1核酸序列分别克隆至含有鞭毛蛋白的质粒p ET42b-Flj B中,构建表达载体p ET/Flj B-AD-2和p ET/Flj B-4×AD-2S1,转化大肠埃希菌Rosetta 2(DE3),IPTG诱导表达,收集Flj B-AD-2的菌体裂解液上清及Flj B-4×AD-2S1的菌体裂解液沉淀,经Superdex-200凝胶过滤层析纯化,根据蛋白含量及纯度配制无佐剂抗原(Flj B-AD-2和Flj B-4×AD-2S1)及含Al(OH)3佐剂抗原[Flj B-AD-2+Al(OH)3和Flj B-4×AD-2S1+Al(OH)3]。用制备的抗原于第0和21 d皮下免疫BALB/c小鼠,设PBS对照组,于2次免疫后1周,眼窝后穿刺采血,分离血清,ELISA法检测AD-2S1特异性Ig G及Ig G1和Ig G2a水平,中和试验检测HCMV中和抗体水平。结果纯化的Flj B-AD-2抗原相对分子质量约54 000,纯度为88.2%,蛋白含量为0.91 mg/ml;纯化的Flj B-4×AD-2S1抗原相对分子质量约50 000,纯度为96.8%,蛋白含量为0.45 mg/ml。与对照组相比,免疫组小鼠均产生不同水平的抗AD-2S1特异性Ig G抗体,Flj B-AD-2和Flj B-AD-2+Al(OH)3免疫组小鼠Ig G水平较低,Flj B-4×AD-2S1和Flj B-4×AD-2S1+Al(OH)3免疫组小鼠Ig G水平较高,其中Flj B-4×AD-2S1与Flj B-AD-2免疫组相比,差异有统计学意义(P<0.05);Flj B-4×AD-2S1可诱导小鼠产生Ig G1和Ig G2a两种抗体,但Ig G2a水平较高,差异有统计学意义(P<0.05),而Flj B-4×AD-2S1+Al(OH)3诱导小鼠产生的Ig G1和Ig G2a水平差异无统计学意义(P>0.05);各免疫组小鼠血清中和抗体水平均不高于1∶4。结论 Flj B-4×AD-2S1和Flj B-AD-2均能诱导产生AD-2S1特异性Ig G,却不能诱导小鼠产生补体非依赖性中和抗体。
Objective To investigate the immune effect of AD2 site Ⅰ (AD-2S1) in mice with human cytomegalovirus (HCMV) glycoprotein B (g B). Methods The AD-2 and 4 × AD-2S1 nucleic acid sequences were cloned into flagellin-containing plasmid pET42b-FljB and the expression vectors p ET / Flj B-AD-2 and p ET / Flj B-4 × AD -2S1 was transformed into Escherichia coli Rosetta 2 (DE3) and induced by IPTG. The supernatant of Flj B-AD-2 lysate and the bacterial lysate of Flj B-4 × AD-2S1 were collected and purified by Superdex -200 gel filtration chromatography to prepare unadjuvanted antigens (Flj B-AD-2 and Flj B-4 x AD-2Sl) and Al (OH) 3 adjuvant antigens [Flj B- AD-2 + Al (OH) 3 and Flj B-4 × AD-2S1 + Al (OH) 3]. BALB / c mice were immunized subcutaneously with the prepared antigens on day 0 and day 21, and PBS control group was established. One week after the second immunization, blood was collected by puncturing the orbital septum, serum was separated and the specific Ig G of AD-2S1 was detected by ELISA Ig G1 and Ig G2a levels, neutralization test to detect HCMV neutralizing antibody levels. Results The purified Flj B-AD-2 antigen had a relative molecular mass of about 54 000, a purity of 88.2% and a protein content of 0.91 mg / ml. The relative molecular mass of purified Flj B-4 × AD-2S1 antigen was about 50,000 with a purity of 96.8%, protein content of 0.45 mg / ml. Compared with the control group, the immune mice produced different levels of anti-AD-2S1 specific Ig G antibody, Flg B-AD-2 and Flj B-AD-2 + Al The levels of Ig G in Flj B-4 × AD-2S1 and Flj B-4 × AD-2S1 + Al (OH) 3 groups were higher than those in Flj B-4 × AD- (P <0.05). Flj B-4 × AD-2S1 induced IgG and Ig G2a antibodies in mice, but the level of Ig G2a was higher, the difference was (P <0.05). There was no significant difference in Ig G1 and Ig G2a levels induced by Flj B-4 × AD-2S1 + Al (OH) 3 in mice (P> 0.05) Mouse serum neutralizing antibody levels were not higher than 1: 4. Conclusion Both Flj B-4 × AD-2S1 and Flj B-AD-2 can induce the production of AD-2S1-specific Ig G, but can not induce the production of complement-independent neutralizing antibodies in mice.