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- Title
CONSTRUCTION AND TRANSFORMATION OF RECOMBINANT PET28A EXPRESSION VECTOR IN BL21 (DE3) CELLS WITH BASIC BIOINFORMATICS ANALYSIS.
- Authors
Al-Muhanna, Sddiq Ghani; Al-Muhanna, Abbas Shakir
- Abstract
The current study re-configured the HMPREF0351_11084 gene (GenBank accession number is NC_017960.1) to incorporate required sequence without impacting the protein structure. Which includes sties for a number of restriction enzymes, 6xHis-tag and stop codon at the end of gene. In addition to the gene optimization sequence through using bioinformatics software, then synthesized and sub-cloned into pGH cloning vector. The synthetic HMPREF0351_11084 gene was cloned into NcoI and EcoRI sites of pET28a expression vector under the control of T7 promoter. The recombinant pET28a expression vector (pET28a-HMPREF0351_11084) was transformed into BL21(DE3) cells. The right transformants of recombinant pET28a construct were verified using colony PCR, then the candidate plasmids (positive colonies) were further analyzed by plasmid PCR, restriction digestion and DNA sequencing. The results of these tests confirm the right construct of recombinant pET28a. To express recombinant target protein, the pET28a-HMPREF0351_11084/BL21 (DE3) cells were cultivated in Luria Bertani broth containing kanamycin antibiotic and induced with IPTG. The protein of expressing bacteria was analyzed by SDS-PAGE and stained with coomassie brilliant blue stain. The stained SDS-PAGE gel shows predicted target protein with molecular weight 24.9 kDa. This recombinant protein maybe serves as a new potential protein vaccine for pneumococcal.
- Subjects
SYNTHETIC genes; GENETIC vectors; RECOMBINANT protein synthesis; SODIUM dodecyl sulfate; POLYACRYLAMIDE gel electrophoresis; BIOINFORMATICS; TYPE specimens (Natural history)
- Publication
Biochemical & Cellular Archives, 2018, Vol 18, Issue 1, p147
- ISSN
0972-5075
- Publication type
Article