家蚕GATA转录因子BmGATA6的克隆、多克隆抗体制备及组织细胞定位

Cloning, Antibody Preparation and Subcelluar Localization of Bm GATA6 in Silkworm(Bombyx mori)

【目的】克隆家蚕(Bombyx mori)GATA转录因子BmGATA6,将其在原核细胞中进行表达,纯化重组蛋白,多次免疫小鼠获得BmGATA6多克隆抗体,为进一步分析BmGATA6在家蚕变态发育中的功能提供抗体条件。【方法】解剖家蚕获得5龄第3天的各组织及4眠至熟蚕整个时期的中肠组织,在液氮中研磨为粉末,利用Trizol法提取RNA,体外反转录得到c DNA。基于家蚕BmGATA6的基因编码序列设计全长特异引物,并以Bm Actin3作为内参,进行家蚕5龄第3天各组织及4龄眠期至熟蚕期各时期的RT-PCR检测。设计原核表达引物,扩增原核表达片段连接到p ET32a载体上,构建原核表达载体,转化至BL21(DE3)Escherichia coil表达菌株。加入IPTG至终浓度分别为0.4、0.6、0.8、1.0 mmol·L^(-1)。在16℃条件下培养20 h和37℃条件下培养5 h分别进行BmGATA6蛋白诱导表达。选取最佳诱导条件进行BmGATA6蛋白的大量诱导,用镍柱亲和层析法纯化家蚕BmGATA6蛋白,经多次免疫昆明小鼠后,最终获得BmGATA6多克隆抗体。利用ELISA法测定鼠抗BmGATA6蛋白多克隆抗体的效价,通过Western blot检测BmGATA6抗体的特异性。以家蚕5龄第6天中肠的石蜡切片作为样本,通过免疫荧光对家蚕中肠组织中的BmGATA6蛋白进行亚细胞定位。【结果】家蚕GATA转录因子BmGATA6的c DNA全长4 011bp,ORF长为1 974 bp,编码657个氨基酸,包括205 bp的5′非翻译区序列(5′UTR)和1 832 bp的3′非翻译区序列(3′UTR)。该基因位于15号染色体上,基因全长12 326 bp,为多外显子基因,共含有8个外显子和7个内含子,具有2个典型的GATA锌指结构域,分别位于第470—521和531—582氨基酸区域。RT-PCR结果显示BmGATA6在5龄第3天家蚕中肠中显著高量表达,其次在马氏管中有少量表达,而在其他组织中没有检测到BmGATA6的表达。同时RT-PCR结果显示BmGATA6在4龄眠期到熟蚕期的中肠组织中持续高表达。构建BmGATA6原核表达载体,通过不同温度与IPTG浓度诱导发现,BmGATA6重组蛋白在16℃诱导主要存在于上清中,在37℃诱导主要存在于包涵体中。在0.6 mmol·L^(-1)的IPTG、16℃下诱导20 h为最佳条件,利用镍柱亲和层析法获得较纯的BmGATA6重组蛋白,并利用纯蛋白多次免疫昆明小鼠制备多克隆抗体,用ELISA法对抗体效价进行了检测,显示BmGATA6抗体效价均高达1﹕128 000。Western blot结果显示BmGATA6抗体能特异地识别家蚕BmGATA6蛋白,其大小约为75 k D,与预测大小相符。免疫荧光结果显示BmGATA6蛋白在家蚕中肠的各类细胞中均有较高表达,且定位于中肠细胞的细胞核中。【结论】成功制备了BmGATA6多克隆抗体,分析了其在中肠组织中的亚细胞定位情况,推测BmGATA6在家蚕中肠的生理变化过程中扮演着重要调控角色,为进一步探索BmGATA6在家蚕中的功能奠定了基础。

【Objective】The objective of this study is to clone and overexpress BmGATA6 in Escherichia coli to obtain the recombinant protein, which can be used to immunize mice for the preparation of polyclonal anti-BmGATA6 antibody. And to provide a theoretical basis for further studying the function of BmGATA6 in silkworm（Bombyx mori）. 【Method】B. mori was killed to obtain the different tissues on day 3 of 5th instar larvae and the midguts from the molting period of the 4th instar larvae to the wandering stage, and these tissues were grinded into powder in the liquid nitrogen. Tissue RNA was extracted by Trizol, and c DNA was reversely transcribed in vitro. The full-length specific primer sequences were designed according to BmGATA6 coding sequence. Using Bm Actin3 as a reference gene, RT-PCR was conducted to detect the expression of BmGATA6 both in the different tissues on day 3 of 5th instar larvae and the midguts from the molting period of the 4th instar larvae to the wandering stage. The prokaryotic expression primer sequences were designed, and the amplified fragment was ligated to the p ET32 a, which was then transformed into BL21（DE3） E. coil to obtained fusion protein. The recombinant BmGATA6 protein was induced with 0, 0.4, 0.6, 0.8 and 1.0 mmol·L-1 IPTG for 20 h at 16℃ or 5 h at 37℃. The optimal induction conditions were chosen to gain BmGATA6 protein, and Ni-NTA affinity chromatography was utilized to purify the BmGATA6 protein. Then the anti-BmGATA6 polyclonal antibody was obtained after immunizing mice with recombinant protein. The expression of BmGATA6 was detected by Western blot with the antibody, which was identified by ELISA. Taking paraffin sections of the midgut on day 6 of 5th instar larvae as samples, the subcellular localization of BmGATA6 protein in the midgut of B. mori was conducted by immunofluorescence.【Result】The c DNA of BmGATA6 was 4 011 bp. The ORF of BmGATA6 was 1 974 bp, encoding 657 amino acids. The c DNA of BmGATA6 was comprised of 205 bp 5′UTR and the 1 832 bp 3′UTR. BmGATA6 was located on chromosome 15 in B. mori genome, and the full-length was 12 326 bp. BmGATA6 was a gene with multiple exons structure, including 8 exons and 7 introns. The predicted protein sequence contained two conserved zinc-finger domains, which were located in 470-521 and 531-582 amino acid regions. RT-PCR results showed that the BmGATA6 was highly expressed in the midguts on day 3 of 5th instar larvae, while the expression was low in the Malpighian tubule, and no BmGATA6 expression was detected in other tissues. Besides, RT-PCR results showed that the BmGATA6 was continually expressed in midguts from the molting period of the 4th instar larvae to the wandering stage. Construction of prokaryotic expression vector of BmGATA6, it was found that the recombinant BmGATA6 protein mainly existed in the supernatant at 16℃, or in inclusion bodies at 37℃. The recombinant BmGATA6 protein was best induced with 0.6 mmol·L^（-1） IPTG for 20 h at 16℃, then purified using Ni-NTA affinity chromatography. To obtain the anti-BmGATA6 polyclonal antibody, mice was immunized repeatedly with pure protein. ELISA indicated that BmGATA6 antibody titer was 1﹕128 000. Western blot demonstrated that anti-BmGATA6 polyclonal antibody, which was prepared by ourself, could detect BmGATA6 protein specifically. BmGATA6 protein molecular weight was about 75 k D, consistent with prediction. Immunofluorescence results showed that BmGATA6 was highly expressed in midgut cells of B. mori, and it was located in nucleus.【Conclusion】Anti-BmGATA6 polyclonal antibody was prepared successfully, and preliminary analysis of its subcellular localization in the midgut tissue was done, which revealed that BmGATA6 played an important role in the regulation of physiological changes in the midgut of B. mori, and laid the foundation for further exploration of the BmGATA6 function in B. mori.