Mechanisms of miRNAs (MicroRNAs) and Their Expression in Gastric Cancer.

Leptospirosis, a significant yet often overlooked zoonotic disease, is distributed worldwide, particularly in tropical and subtropical regions. The manifestation of its symptoms can be deceptive, often leading to misdiagnosis due to its resemblance to other infectious diseases. The development of rapid diagnostic tests and the identification of potential vaccine candidates for leptospirosis pose significant challenges. Surface-exposed antigens, found on the outer layer of Leptospira, likely contribute to the initial interactions between the host and the pathogen.Lipl32 is highly conserved and exclusively produced by pathogenic Leptospires, and it plays a significant role in a prominent immunogen in leptospirosis. The objective of this study is to establish the optimal 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. A comprehensive analysis of all Lipl32 protein sequences was conducted using the NCBI database. The 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. Subsequent purification and confirmation by immune blotting were then performed. BALB/c mice (4-6 weeks old) were vaccinated with three doses containing 50 mg of rLipl32, with a 14-day interval, and compared with controls. The humoral immune response and the cytokine profile were evaluated using an indirect sandwich ELISA test. The results demonstrated that the rLipl32 protein exhibited elevated levels of expression in the presence of 0.5 mM IPTG following a 16-hour incubation period at 22°C. The optimal conditions for the Ni-NTA pull-down process entailed 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. This process successfully purified the rLipl32 protein in soluble form. The administration of rLipl32 resulted in elevated total antibody titers (p<0.05) and a significant increase in cytokine levels (p<0.05). Consequently, rLipl32 was found to potently stimulate specific humoral and cellular immune responses. It has been proposed that this agent could be further utilized for immune dominant Lipl32-based diagnosis and has potential as a subunit vaccine.