Different vectors used to transform and clone of nonstructural NS1 gene of Influenza B in Escherichia coli

Abstract

Flu is a highly contagious and common illness caused by influenza A, B, and C viruses. The aim of the present study was to investigate the transformation and cloning of NS1B gene with pET-32a, pET-32b and pQE-81L in Escherichia coli BL21(DE3) and DH5α. pUC57-NS1B synthetic gene was transform and clone in Escherichia coli BL21(DE3). Isolation, single digestion and ligation with pET-32b using HindIII restriction enzyme. Amplification of recombinant DNA was done with conventional PCR after transformation. Screening with IPTG of colonies. Gel electrophoresis was done for each step of cloning after isolation. Isolation, double digestion and ligation with pET-32a and pQE-81L using SacI, PstI and HindIII respectively.Recombinant DNA was attempted to be transformed into E. coli strains BL21 (DE3) and DH5α. pUC57 plasmid carrying NS1B gene was successful transformed and isolated from E. coli BL21 (DE3). Designed primers used for PCR of NS1B showed successful amplification. First screening of pET-32b-NS1B colonies using white/blue method, cloning NS1B into pET-32b using single restriction digestion with HindIII, pET-32a using double restriction digestion with SacI and HindIII and pQE-81L using double restriction digestion with PstI and HindIII gave unexpected result. This result may relate to re-ligation of digested vector for single digestion and uncompleted digestion for vectors of double restriction digestion. Current study has suggested that recombinant NS1B gene can be cloned using single digestion with other expression vectors.