| 摘要: |
| 【目的】探索快速制备高品质X连锁凋亡抑制蛋白（XIAP）多克隆抗体的方法，为研究XIAP基因的功能及XIAP蛋白在肿瘤组织中的表达量检测提供参考。【方法】通过RT-PCR方法获得人XIAP基因的CDs区序列，与pET151/D-TOPO载体连接，构建XIAP基因的原核表达载体；考察细胞密度（OD600）、IPTG浓度、诱导温度和诱导时间对XIAP重组蛋白表达量的影响，以确定XIAP重组蛋白的最佳诱导条件，并用Ni-NTA亲和层析柱法纯化XIAP重组蛋白；用纯化的XIAP重组蛋白对2只成年新西兰白兔进行常规免疫后，再对其中1只进行耳静脉注射加强免疫，连续3 d，每天免疫1次，用ELISA检测抗体效价，Western blot检测抗体特异性。【结果】①通过RT-PCR方法获得人XIAP基因的CDs区序列，与pET151/D-TOPO载体连接，构建XIAP基因的原核表达载体，测序后，与GenBank中序列的的相似度为97%。② XIAP重组蛋白的最佳诱导条件为：温度30 ℃，初始诱导细胞密度（OD600）为0.6，IPTG 浓度0.5 mmol/L，诱导时间4 h。在此条件下，XIAP重组蛋白多以包涵体的形式存在于细胞沉淀中。③利用Ni-NTA亲和层析柱法，在pH为5.9和4.5的洗脱液中均可获得质量浓度为600 μg/mL的XIAP重组蛋白。④ 耳静脉连续加强免疫是刺激动物机体快速产生抗体的有效方法（31 d）。ELISA 和 Western blot检测结果表明，耳静脉连续加强免疫产生的XIAP抗体的效价和特异性均明显高于常规免疫所产生的抗体。【结论】耳静脉连续加强免疫能够显著提高XIAP抗体的效价，且能有效缩短多克隆抗体的制备时间。 |
| 关键词: 重组蛋白表达 多克隆抗体 XIAP基因 耳静脉连续加强免疫 |
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| 基金项目:西北农林科技大学引进人才启动基金项目(2111020821) |
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| Study on a rapid method for preparation of human XIAP polyclonal antibody |
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| Abstract: |
| 【Objective】This paper aimed to explore the rapid preparation of high-quality XIAP polyclonal antibodies to provide a support for XIAP detection in early diagnosis of tumors.【Method】In the present study,we cloned the CDs fragment of human XIAP gene using RT-PCR technique,ligated with pET151/D-TOPO and constructed the vector pET151-XIAP for prokaryotic expression.Then we systematically investigated the conditions for the high level of the recombinant protein expression in prokaryotic system by testing cell density (OD600),concentration of IPTG,inducing temperature and inducing time.Then we purified the XIAP protein by Ni-NTA kit and immunized rabbait by conventional immunization method coupled with era vein immunization boosting.Finally,we developed a rapid method for preparation of XIAP polyclonal antibody.【Result】①The CDs fragment of human XIAP gene using RT-PCR technique,ligated with pET151/D-TOPO and constructed the vector pET151-XIAP for prokaryotic expression was cloned.The similarity was 97% by sequencing detected.② The optimized expression conditions of XIAP gene were obtained as follows:growth temperature 30 ℃;the initial cell density for induction (OD600) 0.6;IPTG concentration 0.5 mmol/L;induction time 4 h.In this situation,XIAP proteins were in the cell lysis as the Inclusion bodies.③ 600 μg/mL XIAP protein was obtained by Ni-NTA kit.④ Conventional immunization method coupled with era vein immunization boosting for 3 days is an effective way to stimulate animal immune system to produce antibodies.ELISA and Western blot analysis showed that the titre and specificity of serum antibody from optimized immunization method is better than that from conventional immunization method.【Conclusion】Conventional immunization method coupled with era vein immunization boosting could dramatically increase the antibody titres and specificity and significantly shorten the antibody production time. |
| Key words: prokaryotic expression polyclonal antibody XIAP gene immune boosting via era vein injection |
