梅花鹿Thymosin beta 10基因的原核表达、活性鉴定和多克隆抗体制备

Prokaryotic expression,activity testing and polyclonal antibody preparation of thymosin beta 10 gene of sika deer

为研究梅花鹿Thymosin beta 10基因(Tβ10)在鹿茸快速生长过程中的作用,利用Gateway技术构建梅花鹿Tβ10基因的原核表达载体—pDEST17-Tβ10并诱导表达,对融合蛋白进行功能检测及多克隆抗体制备。结果表明:1)获得了190bp Tβ10基因开放阅读框序列,并获得重组表达载体pDEST17-Tβ10。2)pDEST17-Tβ10在宿主BL21-SI中诱导表达条件为0.3mol/L NaCL诱导2h,获得产物的最终浓度为0.50mg/mL。3)制备的Tβ10蛋白多克隆抗体血清效价在105之上,该抗体可以特异性结合天然和原核表达的Tβ10蛋白。4)离体检测发现,Tβ10融合蛋白一方面可以促进人脐静脉内皮细胞增殖,另一方面可以抑制鹿茸前成软骨区细胞的增殖。综上所述,本研究获得了具有生物活性的Tβ10融合蛋白,并制备了高度特异性的梅花鹿Tβ10蛋白多克隆抗体,为深入研究Tβ10基因的生物学功能奠定了基础。

To investigate the role of thymosin beta 10(Tβ10)gene during the rapid growth process of antler,a prokaryotic Tβ10 gene expression vector was constructed by using Gateway technology to test Tβ10 fusion protein activities and produce polyclonal antibody.The results showed that:1)The complete open reading frame(ORF)of Tβ10(190 bp)was inserted into the expression vector pDEST17 via LR cloning reaction which was named aspDEST17-Tβ10.2)The concentration of fusion Tβ10 protein could reach 0.5 mg/mL yielded from host cells BL21-SI under the induction with 0.3 mol/L NaCl for 2 hours.3)The titer of Tβ10 polyclonal antibody researched 105 which could bind to natural or fusion Tβ10 protein in Western blot assay.4)In vitro assay revealed that Tβ10 protein could promote proliferation of HUVEC but inhibited antler pre-cartilaginous cells’.In conclusion,the Tβ10 fusion protein and obtained high titer polyclonal antibody was purified,which would lay a solid foundation for the study of biological functions of Tβ10 gene.