人胰腺核糖核酸酶的基因克隆、蛋白表达与活性测定

Construction of prokaryotic expression plasmid with human pancreatic ribonuclease gene and protein expression and identification

目的:构建人胰腺核糖核酸酶(hpRNase)原核表达载体、获得纯化hpRNase蛋白,为构建乳腺癌单链免疫毒素奠定基础。方法:针对编码hpRNase成熟蛋白基因的cDNA序列设计相应的引物,通过反转录PCR技术,从一例胰腺切除患者的组织标本中,扩增出编码hpRNase成熟蛋白的cDNA序列,亚克隆入表达载体pPROEX-HTb;在大肠杆菌BL21中经IPTG诱导融合蛋白表达,并用镍离子交换树脂进行蛋白纯化;通过RNA水解实验验证hpRNase重组蛋白的活性。结果:经PCR、酶切、测序等方法鉴定,hpRNase cDNA正确克隆入pPROEX-HTb表达载体;IPTG可诱导载体表达分子量约20 kD的蛋白产物,经Western blot验证可与抗hpRNase抗体发生特异性反应;经镍离子交换树脂纯化后的重组蛋白可有效降解RNA。结论:成功构建hpRNase原核表达载体,并获得有RNA降解活性的hpRNase重组蛋白,为进一步构建乳腺癌单链免疫毒素奠定了基础。

Objective： To construct the prokaryotic expression plasmid of human pancreatic ribonuclease （hpR- Nase） gene, and obtain purified active hpRNase protein, in order to construct single - chain immunotoxin of human breast cancer. Methods：The eDNA encoding hpRNase mature protein was amplified by reverse transcription PCR using the RNA template from the specimen of a patient＇s pancreas. The PCR products were subcloned into the fusion expression vectors pPROEX - HTh. After transformed into E coli BL21, fusion proteins were induced by isopropyhhiogalactoside （IPTG）, and purified by Protino Ni - TED Resin. The protein activity was determined by RNA degradation assay. Results：PCR,sequence and restriction digestion results showed that hpRNase eDNA was correctly inserted into pPROEX - HTb. With IPTG induction, a protein with the molecular weight of about 20 kD was observed under the SDS - PAGE analysis, and immunoreactive with anti - RNase antibody reveled by Western blot. After purified by Protino Ni -TED Resin, the protein was proven to be capable to degrade the RNAs. Conclusion：We successfully constructed the expression plasmid of hpRNase, and obtained active hpRNAse protein, which laid a foundation for construction of the single stranded immunotoxin for the treatment of breast cancer.