不同免疫方案制备抗HAb18G/CD147胞外区多克隆抗血清的比较

目的: 制备针对肝癌相关抗原HAb18G/CD147胞外区不同表位的抗血清, 比较不同免疫方案的免疫效果。方法: 以原核表达的GST -HAb18GEF融合蛋白、重组真核表达质粒pcDNA3 /HAb18G及HCC细胞为免疫原, 分别采用蛋白常规免疫、DNA肌肉免疫及pcDNA3 /HAb18G -HCCbooster(DNA -cellbooster)免疫接种BALB/c小鼠。采用间接ELISA和细胞ELISA, 同时测定免疫血清中抗变性和天然HAb18GEFIgG抗体的滴度和Ig亚类。用Westernblot检测各免疫方案制备的抗血清, 与变性HAb18GEF抗原结合的特异性。用免疫荧光法验证DNA- cellbooster免疫接种法制备的抗血清, 与细胞膜上天然HAb18G抗原的结合特异性。结果:以GST- HAb18GEF常规免疫后, 可诱导高滴度的IgG1抗体产生, 但针对的多为HAb18GEF的变性或线性表位; 以pcD -NA3 /HAb18G肌肉免疫后, 可诱导针对其天然表位的IgG2a抗体产生, 但滴度较低; 以DNA -cellbooster免疫后, 可诱导中等滴度、针对其天然表位的IgG2a和IgG1抗体产生。结论: 不同免疫方案可诱导针对HAb18GEF不同表位、不同滴度的多克隆抗血清, 为淘筛针对其不同表位的多样性抗体奠定了基础。

Comparison of polyclonal anti-sera aga-inst extracellular domain of hepatoma associated antigen HAb18G/CD147 prepared by different immunization schemes

AIM: To compare characteristics of polyclonal anti-sera against extracellular domain of hepatoma-associated antigen HAb18G/CD147(HAb18GEF) generated by different immunization schemes. METHODS: BALB/c mice were immunized with GST-HAb18GEF fusion protein expressed in E.coli (routine immunization method), recombinant eukaryotic expression plasmid pcDNA3/HAb18G (intramuscular injection) and pcDNA3/HAb18G plasmid followed by human hepatoma cells booster (DNA-cell booster), respectively. The titers and Ig subclasses of polyclonal anti-sera against denatured and natural HAb18GEF were detected by indirect ELISA and cell ELISA, respectivly. The specific binding of polyclonal anti-sera prepared by different immunization schemes to denatured HAb18GEF was analyzed by Western blot. The specific binding of polyclonal anti-sera produced by DNA-cell booster immunization to natural HAb18G antigen on hepatoma cells was detected by immunofluorescence staining. RESULTS: GST-HAb18GEF immunization could induce polycolonal antibody IgG1 with higher titer mainly against denatured or linear epitopes on HAb18GEF. Antibody induced by pcDNA3/HAb18G intramuscular immunization was IgG2a with lower titer against natural epitopes on HAb18GEF. DNA-cell booster immunization could induce generation of polycolonal antibody IgG2a and IgG1 of moderate titers against the native epitopes on HAb18G. CONCLUSION: The polycolonal sera with different titers against different epitopes on HAb18GEF can be induced by different immunization schemes.