Molecular Cloning And Expression Analysis Of TGF-β1 And TGF-β3 In Half-smooth Tongue Sole(Cynoglossus Semilaevis)

The half-smooth tongue sole(Cynoglossus semilaevis), is an important economic marine fish species in the north of China. With excellent properties such as fast growth,individual large, meat delicious and nutrient-rich, it has become a new kind of excellent breeding object in recent years. But with the enlargement of the farming scale, a variety of infectious diseases become more and more frequently. Especially the bacterial diseases such as the Vibrio harveyi and the Vibrio anguillarum infection, onset fast and spread quickly. These diseases bring great economic losses to the aquaculture industry.In recent years, people use broad-spectrum antibiotics and chemical drugs for fish bacterial disease prevention, control and cure. But these methods can cause pathogenic bacteria drug-resistance increase and the drug residues can pollute the water environment seriously. Therefore, the study of the half smooth tongue sole immune molecular mechanism has been taken seriously and become the hotspot. In this paper,we used molecular biology method and gene engineering technology, carryed out a research about the character and function of two immune related genes in the half smooth tongue sole: TGF-β1 and TGF-β3. We investigated the influence of two genes on the half smooth tongue sole immune regulating mechanism. It has important theory significance to the exploration of the half smooth tongue sole disease-resistant mechanism and breeding. Similarly, the research of TGF-β1 and TGF-β3 recombinant protein expression has important application value for the half smooth tongue sole disease prevention aspects, such as genetic engineering drug for feed additives.First, TGF-β1 and TGF-β3 genes were cloned from liver of the half smooth tongue sole. Sequence analysis showed that TGF-β1 and TGF-β3 amino acid sequences consisted of multiple N-glycosylation sites and a TGF-β family signature. Analysis of phylogenetic tree indicated that TGF-β1 and TGF-β3 of the half smooth tongue sole hada high homology with TGF-β1 and TGF-β3 of other fish. The real-time quantitative PCR expression analysis showed that TGF-β1 and TGF-β3 was expressed in a diverse array of tissues including heart, kidney, intestine, muscle, skin, brain, gill, liver and spleen of healthy half smooth tongue sole and there were significant differences. The highest levels of TGF-β1 were detected in skin, the higher levels were discovered in kidney and the lowest levels were found in muscle. TGF-β3 expressed the highest in skin, higher in brain and the lowest in muscle. After the Vibrio harveyi infection, the expression levels of TGF-β1 showed uptrend in liver, peaked at 48 h after infection,with 3.17 times of the control group, then fell to control levels. In the spleen, TGF-β1and TGF-β3 showed similar expression trend, rising in the first stage and then decreasing. The expression of TGF-β1 and TGF-β3 reached maximum after infection 24 h and 12 h respectively, with 1.70 times and 1.61 times of the control group(P<0.05). In the kidney, the expression trend of TGF-β1 andTGF-β3 were still similar, both peaked at12 h after infection, respectively with 1.23 times and 1.42 times of the control group(P<0.05), then showed a downward trend. In the gill, TGF-β1 expression quantity did not change significantly comparing with the control group. TGF-β3 expression quantity rose in the first stage and then decreased, peaked at 24 h after infection, with 4.71 times of the control group(P<0.05). The above results suggest TGF-β1 and TGF-β3 may play an important role in the immune against bacterial infections in the half smooth tongue sole. This study provided a evidence for the participation of TGF-β1 and TGF-β3 in the immune regulation mechanism in the half smooth tongue sole and also provided a theoretical basis for the half smooth tongue sole molecular immune research.Second, TGF-β1 and TGF-β3 with AvrII and NotI restriction sites were cloned using the method of PCR. Then inserted them into the expression vector pPIC9 K,successfully constructed the recombinant expression vectors. The plasmids were linearized and then were electroporated into Pichia pastoris strain GS115. Colony PCR and sequencing result showed that TGF-β1 and TGF-β3 had been integrated into the genome of Pichia pastoris.By G418 resistance screening of multi copy, we got the multi copy recombinant yeast strains which can grow at 4mg/mL G418 resistance level. The recombinant yeast strains were induced by methanol. The induced expression product was detected and analyzed by SDS-PAGE and Western-blot. Compared with the empty vectors, the TGF-β1 and TGF-β3 recombinant plasmids expression product was found near 45 kD band of the standard protein marker, consistent with the expected size. Itindicated that the TGF-β1 and TGF-β3 get correct expression in Pichia pastoris.In this study, we implemented the recombinant expression of TGF-β1 and TGF-β3 gene in vitro. The recombinant proteins can be directly secreted into the culture medium, it’s very convenient for the later purification and large-scale preparation. This study can provide abundant data for the further study about the function of Cynoglossus semilaevis TGF-β1 and TGF-β3.