Studies On Cloning Of Obese Gene Of Several Different Fish And Expressing Of Cyprinus Carpio Obese Gene

Since the mouse obese(ob)gene and its human homologue were cloned. The ob gene and its function has been become the focus of attention. Leptin was encoded by ob gene and it is an important signal factor to reflect the level of the fat in the body. Leptin also plays an important role in the food intake, energy expenditure, reproduction, immune function, neuroendocrine secretion. Many animals ob gene have been cloned including human, rat and pig. The understanding about obese gene and its function in fish is still very poor. Fish species is numerous. The different species has different feeding-habits, which has affinity with the level of adipose, food consumption and the feeling of satiation. Researching about fish ob gene will be propitious to study the relationship among fat level, food intake, and nerve, endocrine in different feeding-habits fish, which will be benefit to study the molecule mechanism of regulating food intake in fish. Therefore, we attempted to clone obese gene of the different feeding-habits fish and made the research about the characteristic of obese gene expression. It will contribute to the study the function of fish obese gene.1. Molecular cloning of obese gene of several different fishTotal RNAs were extracted from the different feeding-habits fish mesentery adipose tissue, including the polyphagia fish (Cyprinus carpio, Carassius auratus), predatory fish (Mylopharyngodon piceus, Ophicephalus argus, Silurus meridiunlis, Anguilla japonica, Acipenser schrencki), herbivores fish(Ctenopharyngodon idellus, Megalobrama amblycephala) and filter-feeding fish(Aristichthys nobilis, Hypophthalmichthys molitrix). The fish ob gene was amplified by RT-PCR. The product was cloned into vector pMD18-T. Sequence analyzed revealed that the fish ob has a length of 438 nucleotides which encoded a peptide with 146 amino acid residues. The homologue of the nucleotides sequence and amino acid sequence was high up to 99%. Only 2-4 nucleotides have changed among fish. Compared with human, pig and rat, homologous sequence displayed a fairly high degree of conservation. The homologue of the nucleotides sequence were 84%, 86% and 95% and the homologue of the amino acid sequence were 84%, 82% and 96% respectively.2. The expression of carp ob gene in different tissues and different embryo developmental stagesThe RNA of different tissue( liver, adipose tissue, muscle, brain, kidney, heart and spleen) and different developmental stages embryo (Unfertilized eggs, Morula, Blastula embryos, Gastrula embryos, Neurula, Embryos at muscular contraction, Embryos at heartbeat stage, Hatching, Larvae) were extracted. RT-PCR has been performed toanalysis the ob expression. The product of β-actin. was used as control. The result indicated that the carp ob gene was expressed not only maily in adipose tissue and liver but slightly in other tissues such as kidney, spleen, heart, muscle and brain. The ob gene was detected in unfertilized eggs and embryos at different developmental stages. This research can not confirm whether the ob expression in different developmental stages embryos have the developmental changes. The human and rat ob gene was mainly expressed in adipose tissue and placenta. Our results imply that the expression of carp ob gene is different from that of human and mouse ob gene.3. The expression circadian rhythm of carp obese gene and its expression in different physiological conditionsTwo experiments were conducted. Experiment I : carp ob expression in different food intake conditions. Thirty-two carp were divided into four groups of eight. The first group was fed standard diet.The second group was fasted.The third group was fed the high fat diet. The last group was overfeeding. Following 2 weeks the concentration of blood glucose, serum insulin, leptin, triglycerides and cholesterol was measured. The results showed that the serum glucose concentrations of the high fat group were higher than the levels of the control group (p<0.05) and the fasted group (p<0.01).The fasted group and control did not differ from each other. There were no differences in serum cholesterol between the study groups. The serum triglyceride contents of the high fat group were higher than the control group (p<0.01) and fasted group (p<0.01).The serum insulin level in control (p<0.01) and (p<0.01) in fasted group were lower than the high fat groups. The serum insulin level was significantly different between control group and fasted group. The serum leptin concentrations of the high fat group were higher than the control group (p<0.01), fasted group (p<0.01)and overfeeding group(P<0.05). Compared to the control and fasted group, the leptin contents differed each other significantly(P<0.01).The leptin levels of the carp corrected positively with the body weighe(rs=0.817 , P<0.01),the blood glucose contains (rs=0.608 , P<0.01),insulin concentrations (rs=0.831, P<0.01) and triglycerides levels (rs=0.521, P<0.01) .Experiment II. The expression circadian rhythm of carp ob gene. Carp were divided into two groups of ninety six. One group group was fed standard diet, the other group was fed high fat diet for six weeks. Following 6 weeks, blood samples were collected every 2 hours to measure the leptin concentrations. The result showed that the concentration of serum leptin was higher than the normal group (P<0.01). In two groups, the serum leptin levels were highest in midnight and early morning and the lowest around noon. The peak midnight time leptin concentrations were 73.5% and 33.1% higher compared to nadir leptin levels in control group and high fat group respectively. The results indicated thatcarp ob gene expression displayed the circadian rhythm in 24h.4. The expression of carp obese gene in E.coli and the analysis of biological activityRecombinant plasmid pET-28a-ob was constructed to expression ob in E.coli. After induced by IPTG, a high efficiency expression of fusion protein was obtained in BL21.The level of expression was high up to 40% of the total cell protein of E.coli. Molecular weight of expression products is approximately 20 KD by SDS-PAGE which matches the expected molecular weight. Western blotting showed that ob protein takes on strong immunology activity. The fat mouse model was .established. The recombinant leptin was injected to observe the effects of food intake, body weight, body fat and biochemical parameters on fat mouse and normal mouse. Results showed that the recombinant leptin can reduced food intake, body weight and body fat. The serum concentration of glucose, triglyceride, cholesterol, insulin were decreased then leptin injection. The carp leptin can ease the leptin resistance. The weight of body fat correlated positively with the blood glucose contents (rs=0.46, P<0.05), cholesterol concentrations (rs=0.78, P<0.01 ), triglyceride levels (rs=0.767, P<0.01) and insulin contents (rs=0.62, P<0.01) .5. The expression of carp obese gene in Pichia pastorisThe carp ob fragment was inserted into the yeast expression vector pPIC9k containing AOXI promoter and a-factor signal sequence to construct a recombinant plasmid pPIC9K-ob.The transfer vector was transformed into Pichia pastoris GS115 by electroporation. PCR and enzyme cutting confirmed that the gene of interest was inserted into the yeast chromosome. The Transformants were selected by G418. The recombinant strain was induced by methanol. The secreted protein in supernatant was identified using SDA-PAGE. The molecular weight of expressed protein was about 16kD. Western blotting showed that product of expression displayed immunology activity...