斑马鱼心脏标志基因TNC多克隆抗体的制备及表达研究

TNC是心脏发育的标志基因，但该基因在斑马鱼中的表达尚未研究。斑马鱼TNC基因基因的开放阅读框含有5132bp，编码1710个氨基酸，采用生物信息学结合PCR的方法获得了斑马鱼TNC基因的片段。将所得的PCR片段插入原核表达载体pGEX-4T-1中，并将重组质粒（pGEX-4T-1-TNC）转化大肠杆菌BL21；通过IPTG诱导表达GST—TNC融合蛋白，通过尿素洗涤沉淀蛋白并切胶回收纯化融合蛋白，免疫新西兰大白兔制备多克隆抗体。Western blot和免疫组化分析表明，制备的抗体具有良好的高效价性和特异性。利用该抗体进行斑马鱼胚胎抗体染色分析表明，TNC蛋白在心脏组织中特异表达。

Polyclonal Antibody Preparation and Expression of TNC Gene in Zebrafish

TNC is a cardiac marker gene, but the expression of this gene has not been studied in the zebrafish. Ze-brafish TNC gene open reading frame has 5 132 bp, which encodes 1 710 amino acids. A fragment of TNC gene in ze- brafish was cloned by bioinfoimatics combined with PCR technique, and then inserted into pGEX-4T-1 vector. This new reeombined plasmid was transformed into BL21 （E. coli） and expression of the fusion protein was induced with isopropyl- beta-D-thiogalatopyranoside （IPTG）. The GST-TNC fusion protein was purified by UREA and used to immunize white rabbits originated in New Zealand for polyclonal antiserum production. The specificity of prepared antibody was assayed by western blot and confirmed using immunohistochemistry. Western blot and immunohistoehemistry analyses showed that the generated anti-TNC polyconal antibody has high sensitivity and specificity. Use of this antibodies, antibody stai-ning in zebrafish embryos showed that TNC protein is highly expressed in heart tissue.