胰岛激活蛋白B聚体的各亚基克隆及表达的初步研究

Preliminary Study on Gene Clone and Expression About Four Subunits of Islet Activity Protein(IAP) B-oligomer

研究将百日咳杆菌用B-G培养基活化后再在改良的S-S培养基上液体培养,用CTAB-NaCl法提取百日咳杆菌的基因组DNA。经PCR扩增出胰岛激活蛋白B聚体的四个亚基基因,分别将其克隆于pGEX-6p-1载体上,并探讨了在大肠杆菌BL21(DE3)中可溶性表达IAP B聚体各亚基的可能性。通过控制诱导表达温度,S2,S3均可以实现可溶表达,以GST亲和层析得到的S2终产物0.4 mg/L培养物,S3为0.6 mg/L培养物。S4表达虽是可溶性的,但由于融合蛋白的等电点近于7,多步纯化易损失。S5的表达量太低,有待进一步分析。

In this research a Bordet-Gengou agar which contains sheep blood was used to culture the B. pertussis and then transfer single clone to a modified Stainer - Scholte broth to gain pure B. pertussis cells. The genome DNA of B. pertussis was extracted by using a cetryltrimethylammonium bromide （CRAB） - NaCl protocol. The four subunits of IAP B - oligoner were increased and were cloned on a recombinant pGEX - 6p - 1 vector by PCR, respectively. The results show that： S2 and S3 can be expressed in a soluble state and 0.4 mg/L （S2） and 0.6 mg/L （S3） were obtained,respectively after being purified by GST-affinity system; S4 subunit also can be expressed and purified but the yield is low because its being pI is close to 7; but S5 is hard to get the final product.