bamhi hindiii co restricted expression vector pet28a  (New England Biolabs)

Journal: Biology

Article Title: Functional Characterization of MIP_07528 of Mycobacterium indicus pranii for Tyrosine Phosphatase Activity Displays Sensitivity to Oxidative Inactivation and Plays a Role in Immunomodulation

doi: 10.3390/biology14050565

Figure Lengend Snippet: Cloning, expression, purification and Western blot for rMIPPtpB ( A ). ( B ) ( i ) Confirmation of cloning by restriction digestion of the recombinant plasmid containing MIP_07528 and Rv0153c, respectively, ligated in pET28a vector with BamHI and HindIII enzymes. (M) Marker (D) digested plasmid displaying a fallout at the desired gene size of 831 bp of MIP_07528 and Rv0153c, respectively. (U) Undigested recombinant plasmid containing MIP_07528 and Rv0153c, respectively, on a 1% agarose gel ( A ). ( B ) ( ii ) rMIPPtpB and rMTBPtpB protein expression, respectively, via IPTG induction. (M) Marker (U) uninduced culture pellet with no IPTG. (I) Induced culture pellet with 0.1 mM IPTG induction for 20 h at 20 °C, showing the expression of the desired protein ( A ). ( B ) ( iii ) Purification of both rMIPPtpB and rMTBPtpB, respectively, was carried out using Ni-NTA affinity chromatography. (M) marker (P) purified fraction of rMIPPtpB protein. ( C ) Western blot of His-tagged rMIPPtpB and rMTBPtpB left to right: (I) induced cell pellet fraction of rMTBPtpB, (U) uninduced cell pellet fraction of rMTBPtpB, (P) purified protein rMTBPtpB, (M) marker, (U) uninduced cell pellet fraction of rMIPPtpB, (I) induced cell pellet fraction of rMIPPtpB and (P) purified protein rMIPPtpB. The uncropped western blot figures were presented in .

Article Snippet: The amplified PCR products and the pET28a expression vector were digested with BamHI and HindIII restriction enzymes (NEB), followed by ligation using T4 DNA ligase (NEB).

Techniques: Cloning, Expressing, Purification, Western Blot, Recombinant, Plasmid Preparation, Marker, Agarose Gel Electrophoresis, Affinity Chromatography

Journal: Journal of Immunology Research

Article Title: In Silico Design and Characterization of a Multiepitope Vaccine Candidate Against Brucella canis Using a Reverse Vaccinology Approach

doi: 10.1155/jimr/6348238

Figure Lengend Snippet: (A) In silico cloning of the vaccine construct sequence into the pET-28a (+) expression vector. The construct sequence (blue) was cloned between the vector's EcoRI and BamHI restriction sites (black). (B) 1% agarose gel electrophoresis simulation. The lanes show the molecular weight (MW), and the cloning results in positions 1–3, where lane 1, lane 2, and lane 3 represent the vaccine construct (888bp), the pET-28a (+) (5369bp), and the recombinant plasmid (6245bp), respectively.

Article Snippet: The pET-28a (+) expression vector, obtained from the Addgene database ( https://www.addgene.org/ ) [ ], was selected as the cloning vector.

Techniques: In Silico, Cloning, Construct, Sequencing, Expressing, Plasmid Preparation, Clone Assay, Agarose Gel Electrophoresis, Molecular Weight, Recombinant