Design, Development and Immunogenic Evaluation of a Novel Lipl32 Recombinant Protein of Local Pathogenic Leptospira Serovars

Document Type : Original Articles

Authors

1 Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, Ira

2 Department of human vaccines and serum, Razi Institute, Karaj, Iran

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

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

5 Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran

10.32592/ARI.2025.80.1.103

Abstract

Leptospirosis is a significant yet often ignored zoonotic disease found globally, especially in tropical and subtropical regions. Its symptoms frequently lead to misdiagnosis as they resemble those of other infectious diseases. The design of rapid diagnostic tests or detection of possible vaccine candidates against leptospirosis can be challenging. Surface-exposed antigens are located on the outer layer of Leptospira, and these virulence factors likely contribute to the initial interactions between the host and the pathogen.Lipl32 is highly conserved and exclusively produced by pathogenic Leptospires and has a significant role as a prominent immunogen in leptospirosis. The goal of this study is to establish the ideal conditions for the expression and purification of the rLipl32 protein of Iranian pathogenic leptospira and to assess its ability to stimulate cellular and humoral immune responses.
All Lipl32 protein sequences were analyzed from the NCBI database. Codon sequences of serovars were designed and synthesized, and one local dominant Lipl32 pattern was selected after optimization. The construct was sub-cloned into a pET32a+ vector with His-tag and Trx, then transformed into E.coli (BL21) for expression using IPTG. Then purified, and confirmed by immune blotting. BALB/c Mice (4-6 weeks old) were vaccinated with 3 doses containing 50 mg of rLipl32, at a 14-day interval, and compared with controls. The humoral immune response and the cytokine profile were evaluated using an indirect sandwich ELISA test.
Results showed that rLipl32 protein was highly expressed in the presence of 0.5 mM IPTG after 16 hours of incubation at 22°C. The optimal conditions for the Ni-NTA pull-down involved a one-hour binding period at 37°C, followed by five washing steps and the use of an elution buffer with a pH of 7.4 and a 0.3 mM concentration of imidazole and successfully purified in soluble form. The administration of rLipl32 led to strong total antibody titers (p < 0.05) and dramatically increased cytokine levels (P < 0.05). In conclusion, rLipl32 could strongly stimulate specific humoral and cellular immune responses. It has been suggested that it could be further utilized for immune-dominant Lipl32-based diagnosis and has potential as a candidate to be a subunit vaccine.

Keywords

Main Subjects

References

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Archives of Razi Institute
Volume 80, Issue 1
January and February 2025
Pages 103-115

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