CCKBR蛋白的生物信息学分析、原核表达及抗体制备

Bioinformatics analysis,prokaryotic expression and antibody preparation of cholecystokinin type Ⅱ receptor

为了获得胆囊收缩素Ⅱ型受体（CCKBR）的多克隆抗体,建立CCKBR的分子水平检测方法,试验克隆了CCKBR基因CDS全序列并采用生物信息学方法分析CCKBR的抗原表位,根据分析结果合成重组CCKBR基因序列进行原核表达并对表达产物分析鉴定,最后制备多克隆抗体并分析抗体的适用性。结果表明：CCKBR基因CDS全序列扩增成功;抗原区域可能位于35-53,160-175,195-215,245-300,347-368,390-430位氨基酸残基;原核表达的目的条带大小为23.9 ku,与预期结果一致,Western-blot检测条带正确;用包涵体免疫新西兰大耳白兔,获得CCKBR的多克隆抗体,Westernblot检测到CCKBR天然蛋白。说明试验表达CCKBR重组蛋白成功,且由该蛋白制备的抗体能检测出CCKBR天然蛋白,可用于CCKBR的进一步研究。

In order to obtain the polyclonal antibodies of cholecystokinin type Ⅱ receptor（CCKBR） and develop a molecular detection method of CCKBR,the whole CDS sequence was cloned and the epitopes of CCKBR were analyzed by bioinformatics method. Based on the analysis results,the recombinant CCKBR gene sequence was synthesized and the recombinant protein was expressed and identified. Finally,polyclonal antibodies were prepared and the suitability of antibodies was analyzed. The results showed that the amplification of CCKBR CDS sequence was successful. The antigen epitopes region may be located in 35-53,160-175,195-215,245-300,347-368 and 390-430. The purpose band size of prokaryotic expression was 23. 9 ku,which was consistent with the expected results and the band was proved to be correct by Westernblot. The New Zealand white rabbits was immunized by using the inclusion to obtain polyclonal antibodies of CCKBR and the band of natural proteins was proved to be correct by Western-blot. The results indicated that the CCKBR recombinant protein was successfully expressed,and the antibodies prepared from this protein could be used for CCKBR natural protein detection and CCKBR related further research.