EXPRESSION AND PURIFICATION OF rLOA۲۲ OFLEPTOSPIRA INTERROGANS IN PROKARYOTIC SYSTEM

سال انتشار: 1401
نوع سند: مقاله کنفرانسی
زبان: انگلیسی
مشاهده: 167

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شناسه ملی سند علمی:

MEDISM23_403

تاریخ نمایه سازی: 16 مهر 1401

چکیده مقاله:

Background and Aim : Leptospirosis, an overlooked zoonotic infection disease, is a worldwidepublic health problem affecting both domestic animals and humans. Outer membrane proteins ofleptospire are among the most effective antigens which can stimulate remarkable immuneresponses during the infection processes The outer membrane protein A-like protein Loa۲۲ fromLeptospira interrogans is a ۲۲KDa lipoprotein (۱۹۵ aa) with an OmpA domain in the C-terminus.Loa۲۲ is also the first genetically described virulence factor in Leptospira that confirmed by amutagenesis studies. The objective of the present study was expression and purification ofrecombinant Loa۲۲ antigen from Leptospira interrogans in prokaryotic system.Methods : PCR product of Loa۲۲ gene was clonned in E. coli strain plysS using pET۲۳a (+)plasmid as a vector and induced with ۰.۵mM IPTG at ۳۷ °C for ۵ hours. The bacterial pellet wasobtained by centrifugation (۵,۰۰۰ rpm). The resuspended cells were disrupted by sonication. Thecell lysate was solubilized in sample buffer plus ۲ME , Proteins were separated on ۱۲% SDSPAGEand followed by Coomassie Brilliant Blue staining. Expressed protein was purified usingNi-NTA resin and analyzed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) and Western Blotting.Results : The protein was successfully expressed in E.coli plysS strain and purified. the productionof higher level of soluble rLoa۲۲ in native state was at the highest level when post inductionincubation, IPTG concentration, and duration of induction were ۳۷ºC, ۰.۵mM and ۸ h in ۲xTYmedium respectively. SDS-PAGE results showed that the full-length ۳۸kD protein was inducedby IPTG that confirmed with western blot assay.Conclusion : This study is a practical guide to the expression and purification of recombinantloa۲۲ protein for the design of diagnostic kits or vaccine studies based on the recombinant proteinindividually or in combination with other leptospira surface antigens.

کلیدواژه ها:

Loa۲۲ ، expression ، Pet۳۲a(+) ، E. coli strain plysS ، soluble protein

نویسندگان

Mehdi Gharakhani

Department of Microbiology, Faculty of Basic Sciences, Islamic Azad University, Lahijan Branch, Lahijan, Iran. and Department of Microbiology, Razi Vaccine and Serum Research Institute, Agricultural Research, Education and Extension Organization (AREEO),

Pejvak Khaki

Department of Microbiology, Razi Vaccine and Serum Research Institute, Agricultural Research, Education and Extension Organization (AREEO), Karaj, Iran

Mohammad Faezi Ghasemi

Department of Microbiology, Faculty of Basic Sciences, Islamic Azad University, Lahijan Branch, Lahijan, Iran.

Majid Esmaelizad

Department of Biotechnology, Razi Vaccine and Serum Research Institute, Agricultural Research, Education and Extension Organization (AREEO), Karaj, Iran

Majid Tebianian

Department of Immunology, Razi Vaccine and Serum Research Institute, Agricultural Research, Education and Extension Organization (AREEO), Karaj, Iran