利用DNA免疫法制备植物选择标记基因hpt表达蛋白抗体的研究

目的：制备转基因作物中选择标记基因——潮霉素B磷酸转移酶(hygromycin B phosphotransferase，hpt)基因表达产物的多克隆抗体，并探讨影响核酸免疫效果的因素及相应机制。方法：以hpt的cDNA全长插入真核表达载体pcDNA3中，并经限制性内切酶酶切及DNA测序鉴定。以纯化的重组质粒pCDNA3-HPT免疫BALB／c小鼠。用在E．coli中表达并纯化的(His)6—HPT进行ELISA，检测免疫过程中小鼠血清抗体效价的增长状况，并用Western blot检测抗血清的特异性。结果：经3次核酸免疫后，小鼠血清中未发现明显的抗HPT抗体一第4次加强免疫时，将小鼠分为3组，每组两只。第1组改用去内毒素的质粒提取试剂盒提纯的重组质粒免疫，第2组用(His)6-HPT融合蛋白免疫，第3组仍用原来提取的重组质粒免疫。结果发现，第1组小鼠抗血清的效价有所上升，经ELISA检测达1：200；第2组抗血清的滴度显著升高，达到1：2000；而第3组小鼠血清中仍未检测到明显的抗体：Western blot证实，前两组抗血清均可与亲和层析纯化的GST—HPT、(His)6-HPT融合蛋白及其诱导表达的相应菌体总蛋白发生特异性结合。结论：用DNA免疫法成功地制备了抗hpt基因表达产物的特异性抗体，但抗体效价不够理想。推测与hpt基因本身的性质及其在体内表达呈现的水平有关，可望通过州整影响核酸免疫的其他多种因素提高抗体的水平。

Study on the preparing of polyclonal antibodies against plant-selected maker gene hpt expression protein by DNA immunization

AIM: To prepare the polyclonal antibodies (pAbs) against HPT, a kind of plant-selected maker gene encoding protein, by DNA immunization technique and explore the influencing factors for this gene immunization. METHODS: The coding sequence of hpt was cloned into eukaryotic expression vector pCDNA3. The sequence of the plasmid pCDNA3-HPT was demonstrated by restricting enzyme digestion analysis and DNA sequencing. The sequence-correct recombinant plasmids were purified and used to immunize BALB/c mice. The titer and specificity of antisera were detected by ELISA and Western blot, respecitively. RESULTS: No pAb against HPT was detected following three immunization with hpt genes. Then mice were divided into three groups when the forth booster immunization was carried out: 1st group (immunization with the endotoxin-free recombinant plasmids), 2nd group (immunization with (His)(6)-HPT fusion protein expressed in E.coli) and 3rd group (immunization with the same plasmids as before mentioned). As a result, the pAb titer of the 1st group mice increased to 1:200, and the that of 2nd group was up to 1:2 000. Yet the 3rd group detected no anti-HPT antibody. Western blot analysis had proved that antisera of the first two groups could produce specific binding reaction to the purified GST-HPT, (His)(6)-HPT protein and their expressed product (bacterial protein). CONCLUSION: We have got successfully the specific pAb against HPT by DNA immunization, but its titer is yet unsatisfactory, inferring that the character of the hpt gene self and its expression level may play an important role. In order to raise level of serum antibody prepared by DNA immunization, the farther study on various influencing factors still need to be performed.