Identification Of SjTGF-β1 And Study On The Gene Transcription And Expression In The Process Of Liver Fibrosis

Schistosomiasis which is caused by Schistosoma japonicum(S.japonicum) is one of the major parasitic zoonoses. It is harmful for human health. When the hosts such as mouse, human or other mammal animals expose to water containing cercariae, cercariae can drill into host's skin, transform into the schistosomulum and further develop to adult worm. Male and female adult hug together after their sexual organs are mature and then migrate to the portal - mesenteric venous system to settle, mate and lay eggs. A lot of eggs deposite mainly in the intestinal wall and liver tissue. There are 300 ~ 3000 eggs laied by each female a day. It takes 11 days for the ovum in the egg to transform into miracidium and the eggs which contain the miracidium only survival 10 or 11 days. The secretion coming from miracidium penetrates throuth egg shell and causes inflammation and necrosis around the tissue and vascular in liver. Then egg granuloma and liver fibrosis form gradually. Therefore, egg is the main pathogenic factor in chronic, even advanced schistosomiasis.Schistosomes interact with host and utilize many ways to participate immune evasion. For example,molecular / antigen camouflage, simulation, synthesis of neural elements and inhibition of host immune response, etc. Some receptors of Schistosomes distributing on the surface can combind with host's cytokine. Schistosomes can also utilize host's cytokines such as transforming growth factor-β1(TGF-β1)for promoting themselves'development, maturity and reproduction. In a variety of cytokines, TGF-β1 is recognized as the strongest factor for promoting liver fibrosis. The activation of hepatic stellate cell (HSC) is the central link in the process of the hepatic fibrosis. HSCs translate into myofibroblasts and secrete collagen fibers to product collagen and matrix. HSC activated by lipid peroxidation can release a large number of TGF-β1 which in turn cause the activation of HSC and the production of extracellular matrix (ECM). In addition, TGF-β1 can increase plasminogen activator inhibitor producted by sinusoidal endothelial cells, matrix metalloproteinase inhibitors (TIMP) producted by HSC and decrease collagen synthesis protease producted by HSC. All these materials inhibit the normal degradation of ECM. A large number of interstitial collagen and other ECM components deposite in the Disse space and eventually will lead to liver fibrosis. Otherwise, TGF-β1 is also an immune inhibitory factor which plays an immunosuppressive role in the cell-mediated immunity, humoral immunity and non-specific immunity.TGF-βsuperfamily consist of a kind of growth factor peptide subfamily which have similar structure and function. TGF-βis a member of the superfamily, including TGF-β1-6. TGF-β1-3 in mammals have been cloned and expressed. They have a high degree of inter-species homology. TGF-β1 can exist as active or inactive form in the body. The activated TGF-β1 with specific receptors-binding on the cell membrane through the Smad signal transduction pathways play a biological role .Because of its highly evolutionary conservation among the species, we consider that TGF-β1 gene might exist in the S.japonicum and it will play a certain role in liver fibrosis caused by S.japonicum.ObjectiveOwing to S.japonicum-derived TGF-β1 may be play a certain role on promoting hepatic fibrosis, the SjTGF-β1 gene cDNA sequence by RACE method have obtained on the basis of cloning and expressing SjTGF-β1 mature peptide. The focus of this study is on the detection of SjTGF-β1 gene's transcriptional level and protein expression level in different periods of S.japonicum and SjTGF-β1 protein expression level in different stages of infected mice's liver and plasma to confirm the existence of Schistosoma japonicum TGF-β1-like gene primaryly. Methods:1. SjTGF-β1 mature peptide sequence was completely amplificated by Master Zhu Xunmin. SjTGF-β1 gene sequence was obtained by RACE method by Invitrogen Biotechnology Co., Ltd. Homology comparison with the SjTGF-β1 amino acid sequences from other species, topology, hydrophilicity profile, and tertiary structure of SjTGF-β1 were analyzed with tools on bioinformation website or the bioinformatics softwares. After constructing the recombinant plasmid pET-28(+)a- SjTGF-β1 and expressing it in E.coli, the recombinant protein was analyzed by SDS-PAGE and Western-blot.2. SjTGF-β1 gene's transcription level in cercaria, egg, male and female adults of S.japonicum and infected mice's livers in different periods infected with S.japonicum were detected by fluorescence quantitative PCR technique with the special primer pairs.3. TGF-β1 protein in the male and female adults of S.japonicum and mice liver in different period of infected with Sj were detected by Western Blot.4,Using enzyme linked immunosorbent assay (ELISA) method and immunohistochemistry method, the total TGF-β1 expression levels in plasma and liver of mice infected with S.japonicum were analyzed.Results:1. SjTGF-β1 amino acid sequence had 88%, 92% identity with the mouse and human, respectively, especially the functional domain's amino acid sequence (identity were 99% and 100%, respectively). They had a high degree of homology. The protein molecular weight (MW) was 29.8kDa. It was a basic protein because its isoelectric point (PI) was 8.61. It had some hydrophilic linear epitopes similar to mouse's TGF-β1. Using method of retrieval, it was found that the protein structure of SjTGF-β1 had a part sequence of TGF-βpre-peptide and complete sequence of functional domain. Its spatial conformation was similar to TGF-βgene from mouse and human by homology modeling. 2. pET28a(+)-SjTGF-β1 prokaryotic expression vector was successfully constructed and could be induced by IPTG to express the recombinant fusion protein in E. coli BL21 (DE3). The protein mainly existed as a form of inclusion body. Recombinant stains and inclusion body could be both identified by rabbit anti-mouse TGF-β1 polyclonal antibody.3. Compared with the TGF-β1 nucleotide sequence of mouse, the specific primer pairs of SjTGF-β1 were designed in the different region. Taking SjcDNA as template and SjTGF-β1 primer, or taking mouse cDNA as template and mouse TGF-β1 primer, the specific RT-PCR products which were about 250bp could be amplified, respectively. Taking SjcDNA as template and mouse TGF-β1 primer, or taking mouse cDNA as template and SjTGF-β1 primer, there were non-specific bands. It indicated that TGF-β1 gene could be transcripted specially in Sj female and male adults.4. SjTGF-β1 gene was transcripted in different period of Sj, such as cercaria, egg, male and female adults. With the extension of infecting time and chronic disease process, the transcription level of SjTGF-β1 was high during early stage. It reduced at 21d of the infection time and was in the lowest level at 8 week. The transcription level gradually rebounded at 12 week. At the late of stage, it would recover a high transcription level again.5. Western blot results showed that there were target bands existed in male and female adult and the different infected period of mice liver which could be identified by rabbit anti-mouse TGF-β1 polyclonal antibody near the molecular weight about 50kDa. The protein possibly were the potential-related peptide (LAP) of TGF-β1 which had no activity. The protein expression of male adult's is higher than the female adults'and it also was high in later phase of infected mice.6. The total plasma levels of TGF-β1 in infected mice increased with the extension of infection time detected by ELISA.7. The result of immunohistochemitry showed that the normal mice liver tissue had a small amount of expression of TGF-β1. In infected mice liver, the expression level of TGF-β1 was increased with the extension of infection time. On the late stage of infection, TGF-β1's expression level was higher, especially around the egg granuloma and the portal area.Conclusion:1.SjTGF-βgene had a part sequence of TGF-βpre-peptide and complete sequence of functional domain. The length was about 777bp and encoded 258 amino acids. SjTGF-βgene's identity were 89% and 92% with the mouse and human's amino acid sequence, respectively.2. The prokaryotic expressing vectors (pET-28a- SjTGF-β1) was constructed successfully and the recombinant fusion proteins was expressed. The recombinant fusion protein existed mainly in the form of inclusion bodies and could be identified by rabbit anti-mouse TGF-β1 polyclonal antibody. It indicated that SjTGF-β1 protein had immune reactivity.3. TGF-β1 gene could be transcripted specially in eggs, cercaria, male and female adults of Sj.4. The transcription level of TGF-β1 was high in early phase of infection.Then it reduced gradually at the middle phase of the infection and rose in the advanced phase. It showed that SjTGF-β1 might be involved in the process of liver fibrosis and using self-protection to participate in immune evasion.5. One of the different forms of TGF-β1 protein in Sj total protein could be recognized by rabbit anti-mouse TGF-β1 polyclonal antibody. It also showed that SjTGF-β1-like gene expressed in Sj.6. The total expression level of TGF-β1 in plasma and liver of mice infected with Sj increased gradually following infection time's extension. We speculated that the schistosomal hepatic fibrosis might be the bio-cooperation of TGF-β1 which came from both Sj and host.