蛋白质谷氨酰胺酶成熟肽基因mpg原核表达及其多克隆抗体的制备

Prokaryotic expression and polyclonal antibody preparation of mpg, mature peptide gene of protein-glutaminase

蛋白质谷氨酰胺酶(Protein-glutaminase,简称PG)是一种新型蛋白质脱酰胺酶,在蛋白质改性领域具有广阔的应用前景。但是,目前关于蛋白质谷氨酰胺酶的测定方法主要是通过苯酚-次氯酸盐反应测定铵离子含量指示蛋白质谷氨酰胺酶的酶活,该方法精确度和灵敏度有限。因此,利用原核表达系统表达解朊金黄杆菌(Chryseobacterium proteolyticum)分泌的蛋白质谷氨酰胺酶成熟肽基因mpg,纯化后制备其多克隆抗体用于检测解朊金黄杆菌分泌的蛋白质谷氨酰胺酶的含量。首先以解朊金黄杆菌基因组为模板扩增出成熟肽基因mpg,将其与pET32a载体连接构建重组质粒pET32a-mpg,转化至大肠杆菌BL21(DE3)。在25℃条件下,经异丙基-β-D-硫代半乳糖苷(IPTG)诱导6 h,目的蛋白表达量最高、呈可溶性蛋白。通过Ni-NTA柱纯化的重组蛋白mPG-His 6,将其作为抗原,免疫新西兰白兔制备多克隆抗体。间接酶联免疫法检测其效价,所得抗体效价为1∶5 000。蛋白质免疫印迹实验结果说明,该多克隆抗体特异性良好。最后使用该多克隆抗体对解朊金黄杆菌的发酵上清液进行蛋白质免疫印迹反应,检测天然蛋白质谷氨酰胺酶,结果表明该抗体特异性良好,可用于检测解朊金黄杆菌分泌的蛋白质谷氨酰胺酶,为深入研究蛋白质谷氨酰胺酶的生物学功能、建立蛋白质谷氨酰胺酶高产菌株的高通量筛选方法奠定了基础。

Protein-glutaminase(PG)has broad application prospects in the field of protein modification.However,the measurement method of PG is to convert the ammonium ion content in the catalytic reaction to the content of PG by phenol-hypochlorite reaction,which is less sensitivity and accuracy.Therefore,a prokaryotic expression system was used to express the mature peptide gene mpg of Chryseobacterium proteolyticum,and then rabbit polyclonal antibody was prepared after mpg was purified in order to measure the amount of PG produced from C.proteolyticum.First,the mature peptide gene mpg was amplified from the genome of C.proteolyticum,and ligated with pET32a vector to construct recombinant plasmid pET32a-mpg,which was transformed into BL21(DE3).After the recombinant strain was cultured using isopropyl-β-D-thiogalactoside(IPTG)as an inducer,recombinant protein expressed as soluble protein had the highest expression at 25℃for 6 h.The recombinant protein mPG-His6 purified by Ni-NTA column was used as an antigen to immunize New Zealand white rabbits to prepare polyclonal antibodies.The titer of the antibody was 1∶5 000 by ELISA.The results of Western blotting showed that the polyclonal antibody was specific.Through detected the fermentation supernatant of C.proteolyticum using the polyclonal antibody by Western blotting,the results showed that the antibody had good specificity to detect PG produced from C.proteolyticum,which provided the reference to establish high-throughput screening method for high-yielding protein-glutaminase strain.