Earticle

초록

영어

Interleukin-25 (IL–17E) is a novel Th2 pro-inflammatory cytokine belongs to the member of IL–17 cytokine family. In the present study, bioactive recombinant human IL–25 (rhIL–25), the cDNA of mature IL–25 was synthesized using nested PCR and codon bias of prokaryotic host Escherichia coli. The desired template was cloned into the MCS region of expression vector pET28a+. The recombinant vector was transformed into maintenance host Escherichia coli DH5α and the transformants were selected by kanamycin resistance marker. Expression was carried out using IPTG inducible Escherichia coli BL21(DE3) in different media like LB, terrific broth and M9 media. Among all, terrific broth was used for the enhanced production of rhIL–25. SDS–PAGE analysis shows 31 kDa proteins against low molecular weight protein marker. Refolding of inclusion bodies with denaturation buffer (25 mM Tris–HCl [pH 7.2], 5 M Urea, 20 mM β–ME and 200 mM NaCl) yields the rhIL–25 at a concentration of ~ 86 mg/L at 37 0C, where it is high when compared with the expression at 20 0C (~ 16.5 mg/L). Western blot analysis was carried out using anti human IL–17E/IL–25 antibodies. Biological activity of rhIL–25 was determined by the release of IL–6 from PBMC cells. For the first time, under the conditions of current good manufacturing practice (cGMP), bioactive recombinant IL–25 was produced at large scale in soluble form using industrially feasible bacterial host Escherichia coli BL21(DE3).

목차

Abstract
 1. Introduction
 2. Materials & Methods
  2.1. Media, Bacterial Strains and Plasmid
  2.2. Gene Amplification
  2.3. Construction and Expression of Recombinant Human IL-25
  2.4. Inducer Concentration
  2.5. Media Optimization
  2.6. Induction Temperature
  2.7. Purification of Inclusion Bodies
  2.8. Refolded of rhIL-25
  2.9. Western Blot Analysis
  2.10. Biological Activity of rhIL–25
 3. Results & Discussion
  3.1. Construction of Plasmid pET28a+/IL-25
  3.2. Expression of IL-25
  3.3. Effect of Inducer Concentration on Production of rhIL–25
  3.4. Effect of Media Composition on Production of rhIL–25
  3.5. Effect of Post Induction Temperature on Production of rhIL-25
  3.6. Biological Activity Assay
 4. Conclusion
 Acknowledgement
 References

키워드

저자

• Jaya Lakshmi G [ Department of Biotechnology, Acharya Nagarjuna University, Nagarjuna Nagar, Guntur – 522510, Andhra Pradesh, India ]
• Seetha Ram Kotra [ Department of Biotechnology, Acharya Nagarjuna University, Nagarjuna Nagar, Guntur – 522510, Andhra Pradesh, India ]
• JB Peravali [ Department of Biotechnology, Bapatla Engineering College, Bapatla – 522101, Guntur, Andhra Pradesh, India ]
• PPBS Kumar [ Department of Biotechnology, Acharya Nagarjuna University, Nagarjuna Nagar, Guntur – 522510, Andhra Pradesh, India ]
• K Raja Surya Sambasiva Rao [ Department of Biotechnology, Acharya Nagarjuna University, Nagarjuna Nagar, Guntur – 522510, Andhra Pradesh, India ]

참고문헌

발행기관

간행물

표지보기 관심저널 등록

• 간행물명

  International Journal of Bio-Science and Bio-Technology

• 간기

  격월간

• pISSN

  2233-7849

• 수록기간

  2009~2016

• 등재여부

  SCOPUS

• 십진분류

  KDC 505 DDC 605