Volume 22, Issue 3 (2019)                   mjms 2019, 22(3): 135-140 | Back to browse issues page

XML Persian Abstract Print

Download citation:
BibTeX | RIS | EndNote | Medlars | ProCite | Reference Manager | RefWorks
Send citation to:

Rostami B, Irani S, Bolhassani A, Ahangari Cohan R. Expression of the Recombinant Hsp20-Nef Protein for Evaluation of its Immunogenicity against HIV-1 Nef using Indirect ELISA. mjms. 2019; 22 (3) :135-140
URL: http://mjms.modares.ac.ir/article-30-25213-en.html
1- Biology Department, Sciences & Research Branch, Islamic Azad University, Tehran, Iran
2- Hepatitis & AIDS Department, Pasteur Institute of Iran, Tehran, Iran , azam_bolhassani@yahoo.com
3- Pilot Nano-Biotechnology Department, Pasteur Institute of Iran, Tehran, Iran
Abstract:   (6049 Views)
Aims: Nef protein has been considered as an attractive target for the development of therapeutic HIV-1 vaccine. Furthermore, strong immunological properties of heat shock proteins (HSPs) led to their use as for subunit vaccine candidates. In the current study, the generation of Hsp20-Nef fusion protein was performed in E. , and in BALB/c mice.
Materials and Methods: At first, of Hsp20-Nef recombinant protein E. BL21 and Rosetta strains by SDS-PAGE and western blotting using anti-Nef monoclonal antibody. Then, the recombinant protein was purified by a reverse staining method. Finally, its potency was evaluated to elicit antibody response against HIV-1 Nef antigen using indirect ELISA in mice.
Findings: Our data showed a clear band of ~1230bp related to Hsp20-Nef fusion on agarose gel indicating the correct gene cloning in pET28a vector. The expression of Hsp20-Nef protein was confirmed as a clear band of ~47 SDS-PAGE and western blotting. In the immunological assay, the Hsp20-Nef protein and also the Nef protein emulsified with Freund’s adjuvant significantly enhanced the level of total compared to other groups. Moreover, of Hsp20-Nef was higher than Freund’s adjuvant/Nef in protein regimens (p<0.05).
Conclusion: The Hsp20-Nef fusion protein was effectively expressed in E. and significantly induced antibody response against HIV-1 Nef antigen.
Full-Text [PDF 766 kb]   (1600 Downloads)    
Article Type: Original Research | Subject: Molecular Biology
Received: 2018/09/17 | Accepted: 2019/02/7

1. 1- Milani A, Bolhassani A, Shahbazi S, Motevalli F, Sadat SM, Soleymani S. Small heat shock protein 27: An effective adjuvant for enhancement of HIV-1 Nef antigen-specific immunity. Immunol Lett. 2017;191:16-22. [Link] [DOI:10.1016/j.imlet.2017.09.005]
2. Lelièvre JD, Lévy Y. HIV-1 prophylactic vaccines: State of the art. J Virus Erad. 2016;2(1):5-11. [Link]
3. Habibzadeh N, Bolhassani A, Vahabpour R, Sadat SM. How can improve DNA vaccine modalities as a therapeutic approach against HIV infections?. J AIDS Clin Res. 2015;6(4):1000440. [Link] [DOI:10.4172/2155-6113.1000440]
4. Fanales-Belasio E, Raimondo M, Suligoi B, Buttò S. HIV virology and pathogenetic mechanisms of infection: A brief overview. Annali dell'Istituto Superiore di Sanità. 2010;46(1):5-14. [Link] [DOI:10.1590/S0021-25712010000100002]
5. Lever AML. AIDS/HIV, molecular and cell biology. Rev Cell Biol Mol Med. 2006 Sep. [Link] [DOI:10.1002/3527600906.mcb.200300043]
6. Das SR, Jameel S. Biology of the HIV Nef protein. Indian J Med Res. 2005;121(4):315-32. [Link]
7. Geyer M, Fackler OT, Matija Peterlin B. Structure-function relationships in HIV-1 Nef. EMBO Rep. 2001;2(7):580-5. [Link] [DOI:10.1093/embo-reports/kve141]
8. Lema D, Garcia A, De Sanctis JB. HIV vaccines: A brief overview. Scand J Immunol. 2014;80(1):1-11. [Link] [DOI:10.1111/sji.12184]
9. Wallin RP, Lundqvist A, Moré SH, Von Bonin A, Kiessling R, Ljunggren HG. Heat-shock proteins as activators of the innate immune system. Trends Immunol. 2002;23(3):130-5. [Link] [DOI:10.1016/S1471-4906(01)02168-8]
10. Wan T, Zhou X, Chen G, An H, Chen T, Zhang W, et al. Novel heat shock protein Hsp70L1 activates dendritic cells and acts as a Th1 polarizing adjuvant. Blood. 2004;103(5):1747-54. [Link] [DOI:10.1182/blood-2003-08-2828]
11. Bakthisaran R, Tangirala R, Mohan Rao C. Small heat shock proteins: Role in cellular functions and pathology. Biochimica et Biophysica Acta Proteins and Proteomics. 2015;1854(4):291-319. [Link] [DOI:10.1016/j.bbapap.2014.12.019]
12. Simpson RJ. Zinc/Imidazole procedure for visualization of proteins in gels by negative staining. CSH Protoc. 2007;2007:pdb.prot4701. [Link] [DOI:10.1101/pdb.prot4701]
13. Alipour Z, Eskandari N, Mokhah S. Evaluation of knowledge and attitude of non-medical students about AIDS. J Holist Nurs Midwifery. 2016;26(79):10-20. [Persian] [Link]
14. Kadkhodayan S, Irani Sh, Sadat SM, Fotouhi F, Bolhassani A. Cloning, expression and purification of the recombinant HIV-1 Tat-Nef fusion protein in prokaryotic expression system. Arak Med Univ J. 2016;19(4):60-8. [Persian] [Link]
15. Skwarczynski M, Toth I. Peptide-based synthetic vaccines. Chem Sci. 2016;7(2):842-54. [Link] [DOI:10.1039/C5SC03892H]
16. Norimine J, Mosqueda J, Palmer GH, Lewin HA, Brown WC. Conservation of Babesia bovis small heat shock protein (Hsp20) among strains and definition of T helper cell epitopes recognized by cattle with diverse major histocompatibility complex class II haplotypes. Infect Immun. 2004;72(2):1096-106. [Link] [DOI:10.1128/IAI.72.2.1096-1106.2003]
17. Bandehpour M, Sharifnia Z, Mosaffa N, Kazemi B, Seyed N, Soleimani Darani M. Effect of HSP90 adjuvant on immunogenicity of HbsAg in BALB/c mice. Res Med. 2013;37(2):80-4. [Persian] [Link]
18. Matsushima-Nishiwaki R, Kumada T, Nagasawa T, Suzuki M, Yasuda E, Okuda S, al. Direct association of heat shock protein 20 (HSPB6) with phosphoinositide 3-kinase (PI3K) in human hepatocellular carcinoma: Regulation of the PI3K activity. PLoS One. 2013;8(11):e78440. [Link] [DOI:10.1371/journal.pone.0078440]

Send email to the article author

Rights and permissions
Creative Commons License This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.