| CS5931 was derived from Ciona savignyi. It possesses potent antitumor activity and its molecular weight is 5931Da. Study in our laboratory has shown that CS5931 can inhibite the growth of many kinds of tumor cells and induced apoptosis in human hepatoma cell line BEL-7402 in vitro. N-terminal amino acid sequence determination of CS5931 indicated that the polypeptide displays high homology with the sequence of human granulin(GRN). In the present study, the full length cDNA of CS5931 precursor was cloned by RACE (rapid amplification of cDNA ends) approach and the amino acid sequence of CS5931 was obtained, and the polypeptide was expressed in E. coli with high efficiency. In our study the expressed protein was purified through affinity chromatography. The antitumor activity and molecular mechanism of recombinant CS5931 will be researched later.The gene fragment coding CS5931 was successfully cloned and then linked into prokaryotic expression carrier pET21a(+). The expression vectors pET21a(+)-CS5931 was transformed into E. coli Origami (DE3) with chaperone plasmid pG-KJE8 to construct protein expression system Origami/pET21aCS5931+pG-KJE8. Then recombinant polypeptide CS5931-His was induced and expressed successfully in soluble form using Origami/pET21aCS5931+pG-KJE8. After denaturation, purification, renaturation and digestion with protease, we get the recombinant polypeptide CS5931-His with His-tag and its purity was above 95%.Recombinant polypeptide CS5931-His with His-tag in the end of the C mainly expressed in soluble form. This study suggests that we can obtain the purified recombinant polypeptide CS5931-His by collecting soluble protein (supernatant fluid of cracking bacteria) under the condition of denaturation, purification with the nickel ion affinity chromatography column, washing miscellaneous protein with the concentration of 50 mm imidazole, elution with the concentration 300 mm imidazole.Antitumor effect of CS5931-His was studied by analysis of cytotoxicity activity of tumor cells in vitro by MTT assay. The results showed that CS5931-His significantly inhibited the growth of the selected five cells, including human cervical carcinoma Hela cells, human colon carcinoma HCT116, RKO cells, human A549 lung carcinoma cells, human leukemia HL60 cells. The study is important for the development of the polypeptide as novel anticancer agent. The study also provides insight into the understanding of the function of Enolase 1 as a novel target in cancer treatment.In march,2014, the central committee submitted the proposal on the 12th sessions of CPPCC about using of non-food crops repair heavy metal polluted soil. It is said in the proposal, The situation of the heavy metal pollution of soil is becoming more and more serious in our country. We can use non-food crops such as economic crops of cotton, ramie, industrial oil crops castor, sea kale, etc., and other non-food crops move it out of the soil by absorbing the enrichment of heavy metals, to achieve the purpose of pollution control and ecological restoration. It has obvious advantages in repairing effect and economic benefit, and both economic and environmental benefits at the same time, so it’s more acceptable and promotion.Cotton is one of the major crops in the world. It has high yield and low production cost. Cotton’s planting area in China is about 5 million hectares (75 million acres), the output is around 7 million tons.The Yellow River, the Yangtze river basin and the northwest inland cotton region is China’s three major recorded, including 13 provinces such as hebei, shandong, henan, tianjin, jiangsu, anhui, hunan, hubei, jiangxi.Therefore, we choose cotton to process the heavy metal pollution of soil.Our work aims at transing the ACBP1 gene come from the university of Hong Kong into agrobacterium LBA4404, then transduce the gene into the cotton using the method of agrobacterium mediated. So to increase the heavy metal resistance of cotton greatly and control the situation of the heavy metal pollution of soil.The experiment mainly explores the agrobacterium mediated method, and the establishment of the regeneration system of cotton.Experiment select "stone of 434" and "greatness" stone "miscellaneous" three kinds of cotton, finally selected to "stone" of hypocotyl as gene transformation receptor. To optimize the tissue culture conditions, screening of high rate of embryonic callus out the condition, in view of the specific needs of the callus culture, examines the ratio of sucrose, AGAR and different hormone, adding anti Browning agent and other factors, then optimize the suit "stone of 434" genetic transformation regeneration system.Transe the ACBP1 gene into cotton. |
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