Production and characterization of recombinant human leukemia inhibitory factor and evaluation of anti-fertility effects of rabbit anti-rhLIF in Balb/c mice
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Fateme Zare,1 Erfan Zaker,2 Seyed Mohammad Seifati,3 Ali Akbar Saboor-Yaraghi,4 Mahdi Dehghan-Manshadi1,5 Mohammad Mehdi Amiri,6,*
1. Reproductive Immunology Research Center, Shahid Sadoughi University of Medical Sciences, Yazd, Iran
2. Department of Medical Genetics, Faculty of Medicine, Shahid Sadoughi University of Medical Sciences, Yazd, Iran
3. Reproductive Immunology Research Center, Shahid Sadoughi University of Medical Sciences, Yazd, Iran
4. Department of Immunology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran
5. Reproductive Immunology Research Center, Shahid Sadoughi University of Medical Sciences, Yazd, Iran
6. Department of Immunology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran
- Introduction: Human leukemia inhibitory factor (hLIF) is a cytokine of interleukin-6 family. This study aimed to evaluate the recombinant production rate of active hLIF by different vector-host systems under various conditions. Moreover, a rabbit polyclonal antibody (pAb) against recombinant hLIF (rhLIF) was produced and its anti-fertility effects were explored in Balb/c mice.
- Methods: Four different constructs including pET22b/hLIF, pET28b/hLIF, pET32b/hLIF and pColdI/hLIF were designed and transformed into BL21-(DE3), Rosetta-(DE3), Origami-(DE3) and Shuffle T7-(DE3) host cells. The expression level and proliferative effect of rhLIF were measured by SDS-PAGE and MTT assays, respectively. Rabbit pAb to rhLIF was produced and characterized using enzyme-linked immunosorbent assay and western blot techniques. The Balb/c mice were divided into two intervention and control groups. Then, they were intraperitoneally injected by purified rabbit anti-rhLIF and non-immunized rabbit pAb, respectively. After sacrifice on day 7, the number of implantation sites was counted.
- Results: The rhLIF was successfully expressed by pET32b/hLIF and pColdI/hLIF vectors in all hosts with no significant difference in the rate of their expression. The rhLIF was purified and checked for activity. The results showed that it is functionally active and the produced anti-rhLIF pAb could specifically bind to commercial rhLIF. Passive immunization results showed that anti-rhLIF antibody completely inhibited fertility in all injected Balb/c mice compared to controls
- Conclusion: Although previous studies showed expression of rhLIF using various methods, using different vector-host systems ensures us of successful biological active expression of it. The pAb against rhLIF could be a powerful tool for inducing in vivo infertility.
- Keywords: Leukemia inhibitory factor, Anti-ferti, Recombinant protein, pET32b/hLIF vector, pColdI/hLIF vector
