The Fermentation And Expression Of NTL, A Lectin From Chinese Narcissus(Recombinant Narcissus Tazetta Var.Chinensis Roem),in The Yeast Pichia Pastoris

Chinese Narcissus lectin (NTL) has high medicinal value. According to research, NTL has a variety of pharmacological activities, such as immunomodulatory activity for fungi, mammalian cell, against HIV-1virus activity, anti Herpes simplex virus and cytomegalovirus activity, and so on. However, limiting by the sources of raw materials, China Narcissus lectin has a low yield. While, as prokaryotes, like E. coli, express NTL, the results show that a large part of the recombinant protein is present in the form of inclusion bodies, no biological activity, so this topic using recombinant pichia expressn Chinese narcissus lectin.This topic early use Pichia pastoris secretory expressing NTL, although the product after the separation and purification has a certain blood coagulation activity, the yield of target protein can not be effectively measured. In addition, the blood coagulation activity of this product remains low compared with the natural product. Therefore, it is not conducive to large-scale production.This study firstly use Balb/C mice made the Chinese Narcissus lectin polyclonal antibodies to create a method of ELISA, that can efficient, rapid detect recombinant Chinese narcissus lectin from fer mentation broth. Meanwhile, the amount of antigen was determined for each hole10μg, and the best detection condition is primary an tibodies’dilution ratio1:1600, second antibodis’dilution ratio1:150. Then, screening the strain with higher yield and good coagulation effect from the laboratory pre-constructed two strains, GS115/pPIC9K-NTL1and GS115/pPIC9K-NTL2. Finally, determine GS115/pPIC9K-NTL2as a genetically engineered strain of bacteria.Optimize Pichia expression conditions by shake flask experiments. The best condition is to use inorganic salt medium, the cell growth phase bottled liquid proportion control in10%, the initial inoculum is5%, the optimum temperature is30℃, the optimum pH is4.5; during the induction phase of the cell expressing the optimum temperature is28℃, the optimum pH value is5.0, the methanol concentration is1%. Meanwhile, compared to the impact of the three ways to induce the expression of the target protein, the experimental results show that the double carbon alternating stimulation obtain higher yields and protein coagulation activity. Use5L fermenter fermentation expression system to study, investigate the basic conditions of the shake flask select the optimal conditions to study the fermentation process of dry cell weight, changes in glycerol concentration, salt concentration, the concentration of Chinese narcissus recombinant lectins and changes in coagulation activity. The experiments also compared two strategies to induce expression yield of the recombinant protein and coagulation activity.Finally, with hollow fiber membrane ultrafiltration and ultrafiltration at the early stage, the concentration of fermented liquid enrichment ratio is58times and10times respectively. Meanwhile, with molecular sieve chromatography, hydrophobic chromatography and affinity chromatography media mannan concentrated product for further separation and purification, purification factor were obtained13and30respectively. At last, with the Western-Blot purified proteins specifically identified, it confirmed that the product purified is recombinant Chinese narcissus lectin.