Cloning recombinant consensus interferon with overlap extension polymerase chain reaction

Shun Ai Liu, Ryutaro Asano, Jing Jing Guo, Zhi Ying Liu, Kang Kang Yu, Dao Zhen Xu

Research output: Contribution to journalArticlepeer-review

1 Citation (Scopus)

Abstract

AIM: To obtain the recombinant consensus interferon (cIFN) gene by overlap extension PCR and to express it using a prokaryotic expression system to identify its activities. METHODS: Overlap extension PCR was performed to obtain the cIFN gene and to construct the high expression vector for the production of cIFN protein according the preferred codon of E.coli. The expression vector pRA-cIFN was transformed with BL21 (DE3) E.coli straining. The expression of cIFN was identified by SDS-PAGE and Western blotting and purified by metal-chelating chromatography, and refolded by stepwise dialysis method. Then, the immune and biological activities of cIFN were detected by ELISA, MTS chromometry and hemagglutination inhibition test. RESULTS: The cIFN gene was obtained by overlap extension PCR. Its DNA length was 534 bp which is consistent with its theoretical length. SDS-PAGE and Western blotting showed that the pressed cIFN protein in E.coli BL21 (DE3) was in the form of inclusion bodies and its molecular weight was 23.3 kDa. We achieved the refolding of E.coli-expressed cIFN with a step-wise dialysis method. The biological activity of the refolded cIFN was detected by ELISA, MTS chromometry and hemagglutination inhibition test, showing that cIFN had obvious immunological activities, dose-dependent suppressive activity of PLC/PRF/5 cell proliferation and HBsAg secretion inhibitory activity. CONCLUSION: Overlap extension PCR is a simple method to obtain the recombinant genes. cIFN protein produced in this study has significant anti-virus and anti-tumor cell proliferation activities.

Original languageEnglish
Pages (from-to)479-483
Number of pages5
JournalWorld Chinese Journal of Digestology
Volume16
Issue number5
DOIs
Publication statusPublished - 2008 Mar

Keywords

  • Activity identification
  • Overlap extension polymerase chain reaction
  • Prokaryotic expression
  • Recombinant consensus interferon

ASJC Scopus subject areas

  • Gastroenterology

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