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Abstract：
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. 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. 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. 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.
摘要：
人白血病抑制因子(hLIF)是白细胞介素-6家族的细胞因子。本研究旨在评估不同载体-宿主系统在各种条件下活性hLIF的重组生产率。此外，制备了针对重组hLIF（rhLIF）的兔多克隆抗体（pAb），并在Balb/c小鼠中探索了其抗生育作用。四种不同的构建体，包括pET22b/hLIF，pET28b/hLIF,pET32b/hLIF和pColdI/hLIF被设计并转化为BL21-(DE3)，Rosetta-(DE3),折纸-(DE3)和ShufferT7-(DE3)宿主细胞。SDS-PAGE和MTT法检测rhLIF的表达水平和增殖效应,分别。使用酶联免疫吸附测定和蛋白质印迹技术产生并表征rhLIF的兔pAb。将Balb/c小鼠分为干预组和对照组。然后，用纯化的兔抗rhLIF和未免疫的兔pAb腹腔注射，分别。在第7天处死后,计数植入部位的数目。通过pET32b/hLIF和pColdI/hLIF载体在所有宿主中成功表达rhLIF，其表达率没有显着差异。纯化rhLIF并检查活性。结果表明，它具有功能活性，所产生的抗rhLIFpAb可以特异性结合商业rhLIF。被动免疫结果显示,与对照相比,抗rhLIF抗体完全抑制所有注射的Balb/c小鼠的生育力。尽管以前的研究表明rhLIF的表达使用各种方法，使用不同的载体-宿主系统确保我们成功的生物活性表达它。抗rhLIF的pAb可能是诱导体内不育的有力工具。
