Preliminary Study On Gene Clone And Expression About Four Subunits Of Islet Activity Protein (IAP) B-oligomer

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 (CTAB)-NaCl protocol.The individual B oligomer subunits gene(S2～S5) were obtained by PCR. In this process, the method of nest PCR was used to distinguish the S2 gene and S3 gene. S4 and S5 gene were acquired by normal procedure. All four genes were directly cloned into pGEX-6p-1 after digested by BamH I and Xho I. The final recombinant have been sequenced and the results show their sequences are identical to B. pertussis strain Tohama I.After transduction four recombinant vector into E. coli BL21(DE3), the inducing temperature were tested at three stage 14℃, 20℃and 25℃, all four GST-fusion subunits expressed in a low soluble state at 20 and 25℃, but S2 and S3 can express in high soluble ways at 14℃and final 0.4 mg/L of S2 and 0.6 mg/L of S3 were gained by GST-Affinity chromatograph. The further studies should be done for enhancing the yield of S2 and S3 and finding a new way to express the S4 and S5.