Characterization, Soluble Expression And Biological Activity Of Leukemia Inhibitory Factor (lif) From Nile Tilapia

Leukemia inhibitory factor (Lif) was separated and purified from a murine Krebs ascites cells in 1988. It is named because it can induce the leukemia cell M1 to differentiate into the normal cell. It belongs to the interleukin (IL)-6 sub-family. IL-6 sub-family plays an important role in the formation of the hemocyte, inflammatory response and the immune response. Many reports show that Lif has diverse biological functions in mammals and it plays an important role in survival, formation and repair of the neurons, blood generation, hormone production and embryonic development and so on. In recent years, Lif is widely used in clinical and scientific research and it has huge market demand. In clinic, Lif is used to prevent the neuropathy caused by the chemotherapy, promote the implantation of the transplanted embryonic of the infertile women; in scientific research, it is widely used in the culture of the neural stem cell, ESC and IPS to maintain the undifferentiated state, and it takes 90% cost of the stem cell culture. Therefore, how to obtain Lif with biological activity is extremely important. At present, the main problem of Lif prokaryotic expression is mainly in the form of inclusion body, which is difficult to obtain biological activity. In view of this, this paper takes tilapia (in this paper, if not stated, tilapia is designated as Nile tilapia) as the object, the lif was separated and identified from genome and gonadal transcriptome. Moreover, its embryonic development and tissue expression pattern were observed by RT-PCR; the recombinant expression vector pSUMO-ntLif32-221, which expresses the target protein ntLif32-221 with a recombinant protein tag His6-SUMO, was reconstructed, and then induced under different concentrations of IPTG, temperatures and time duration. The soluble target protein could be obtained after induction and there will be a preliminary study of its biological function.On the other hand, fish gonadal cells contain somatic cells and germ cells, which the somatic cells, including ovarian granulosa cells, theca cells, stromal cells, and testicular Sertoli cells, Leydig cells, since the world’s first cell lines RTG-2 derived from rainbow trout(Oncorhvnchus mykiss) gonad was established, currently only a few, such as medaka, flounder, Southern catfish gonadal cell lines have established.Tilapia is a good animal model for studies of sex determination and differentiation in fish.The establishment of gonadal cell lines provids a powerful tool for the research of germ cell proliferation and differentiation, gene functions. So far, there are no tilapia gonadal cell lines. Based on this, the study also conducted testicular, ovarian somatic and germ cells from tilapia in vitro culture conditions and initial exploration of germ cell transplantation. The main findings are as follows:1 Isolation and identification of Tilapia leukemia inhibitory factor(ntlif) cDNA, soluble expression and biological activity1.1 Through bioinformatics analysis and RT-PCR, the cDNA of leukemia inhibitory factor (lif) was cloned from Nile tilapia (Oreochromis niloticus), which contains a 5’ UTR of 153 bp, a 3’UTR of 248 bp and an open reading frame of 666 bp, encoding 221 amino acids. There are a predicted signal peptide within the 1-31 residues, two potential N-glycosylation sites and 4 cysteine residues, which are similar to the typical motifs in mammals. Phylogenetic analysis suggests that the ntLif is a true ortholog of mammalian Lif/OSM. Multiple amino acid sequence analysis showed that ntLif have consistency of more than 35.5% with other fish, while mammals amino acid identity of the Lif only about 18.0 percent, less conservative, Lif possess a species-specific genes. Tilapia Lif protein molecular theory 21.294 KDa, isoelectric point of 8.83.1.2 The mRNA expression of ntlif was observed by RT-PCR throughout the embryo development (from 12 h to 5 d after fertilization) till adult, with highest expression in the heart, spleen, intestine and muscle. Lif may play a biological effect in tilapia among process of embryonic development.1.3 The recombinant expression vector pSUMO-ntLif32-221, which expresses the target protein ntLif32-221 with a recombinant protein tag His6-SUMO, was reconstructed, pSUMO-ntLif32-221 was transfected into E.coli BL21 (DE3), and then induced under different concentrations of IPTG, temperatures and time duration. The soluble target protein could be obtained after induction by 0.2 mM IPTG at 16 ℃ for 20 h. Through SDS-PAGE and Western blot detection, two specific bands of the molecular weight 37 kD and 25 kD were observed, which were compatible with the predicted sizes of the recombinant protein and purified ntLif32-221, respectively. On average, about 10 mg ntLif32-221 were obtained from 1 L-cell cultures after Ni-NTA affinity chromatography and ULP1 digestion.1.4 The detection of the purified protein ntLif32-221 function in proliferation activity of medaka embryonic stem cells (MESl)have taken by FACS, CCK8 assay, the results show that the purified protein ntLif 32-221 promote embryonic stem cell proliferation.2 Tilapia gonadal cell culture and germ cell transplantation2.1 The testis and ovary from 3 months tilapia was dissociated with collagenase IV and trypsin-EDTA solution. The results show that the testis and ovarian cells maintain good growth state in L-15 medium supplemented with 25 mM Hepes,15% FBS,500 U/mL penicillin and streptomycin,1 ng/mL leukemia inhibitory factor (Lif),1% non-essential amino acid,0.5 mM β-mercaptoethanol and 5% autologous serum.2.2 The testis and ovarian cells began to fibroblast-like growth when cultured for 2-3 days in L-15 medium, about 15 days later covered with a single layer, with 0.25% trypsin-EDTA digestion,1:2 subculture, the primary passage of cell growth is relatively slow with an average of 7-10 days for passage.After 9th passage, cell growth began to accelerate with an average 3-6 days for passage. After 120d subculture, the testis cells have spread to the 14th, the ovarian cells spread to 12th, which were named for NTT, NTO.2.3 In the L-15 conditioned medium, tilapia germ cells are not adherent or adherent strong, thus significantly different from somatic cells, the first week was observed spermatogonia, oogonia kind of cell proliferation,swimming sperm have been observed in 3 weeks, thus suggesting that tilapia germ cells can be efficiently cultured in vitro, and complete the spermatogenesis.2.4 Adult female Nile tilapia (XX) with treatment of Busulfan and cyclophosphamide as the recipient, Hoechst33342-labeled germ cell suspension from fish male (XY) were microinjected in the recipient through gonopore, the detection of gonad in 20 days through RT-PCR,but the results failed to amplify the Y chromosome specific molecular markers.The study acquires soluble purified protein for the first time, which lay an important foundation for the biological function in lower vertebrates. Furthermore, the preliminary study of gonadal cells culture of tilapia provides an important tool for the research of germ cell proliferation, differentiation and gene function.