人C型溶菌酶发酵与分离纯化工艺的研究

[目的]研究毕赤酵母菌株(Pichia GS115)表达人C型溶菌酶(H-LYZ)的发酵与分离纯化的工艺.[方法]探讨酵母生长规律,研究不同菌体浓度、不同诱导时间对蛋白产量的影响,并且探讨不同pH条件对DEAE-SepharseFF和CM-Sepharose FF层析分离纯化目标蛋白的影响.[结果]毕赤酵母菌株表达H-LYZ的最佳发酵条件为:发酵24h用浓度1％甲醇诱导,以后每12 h用浓度1％甲醇诱导,共6次,在此条件下可获得高产量目标蛋白.DEAE-Sepharose FF和CM-Sepharose FF分离纯化目标蛋白的最佳pH分别为6.65和6.50.[结论]该研究可为人C型溶菌酶的工艺化生产提供理论指导.     

响应面法优化重组毕赤酵母菌表达β-甘露聚糖酶的诱导条件

[目的]对重组毕赤酵母菌表达β-甘露聚糖酶的诱导条件进行优化。[方法]以重组毕赤酵母GS115为研究对象,通过单因素试验确定最佳产酶条件,并采用响应面试验确定3个因素的最佳水平。[结果]最佳产酶条件为:培养温度28℃,培养pH 6.5,摇床转速210 r/min,培养基装液量100 ml。响应面试验确定3个因素即装液量、甲醇添加量和pH最佳值分别为103.18 ml、1.77%和6.69;在此条件下,β-甘露聚糖酶的活力最大值为4 917.5 U/ml。[结论]验证试验证明模型预测值准确、可靠,为重组毕赤酵母菌的合理开发利用提供了依据。     

Gene cloning of porcine adiponectin gene from adipose tissue and construction of its eukaryotic expression vector

To clone adiponectin (ADPN) gene from Shaziling porcine adipocyte and construct its eukaryotic expression vector, total RNA was extracted from subcutaneous fatty tissue. One pair of specific primers was designed by Primer 5.0 software according to the sequence of ADPN gene of porcine available in GenBank. The ADPN gene was amplified by PCR from cDNA and cloned into pMD18‐T vector to construct recombinant clonal vector pMD‐ADPN, sequenced and analysed. A recombinant expression plasmid pPICZaA‐ADPN was constructed by subcloning the cloned ADPN gene into the linearized pPICZaA vector. Then, the plasmid pPICZaA‐ADPN was expressed in Pichia pastoris (GS115) by electrotransformation. Western blot and Bradford analysis were used to determine the target protein induced by methanol. Results showed that the genome size of ADPN was 732 bp and encoded 244 amino acid, the nucleotide sequence of ADPN shared 100% identity with that of porcine available in GenBank. Western blot and Bradford analysis showed that the recombinant ADPN was expressed in GS115 correctly and has certain immune activity. The expression level of ADPN was 28.5 μg/ml. In conclusion, the recombinant ADPN could express in eukaryotic expression vector pPICZaA‐ADPN constructed in this study effectively.     

抗速灭威单链抗体基因在巴斯德毕赤酵母中的表达及性质研究

选用巴斯德毕赤酵母（Pichia pastoris）系统表达特异性抗速灭威单链抗体（scFv）基因,以为速灭威特异性抗体的大量制备奠定基础。设计引物扩增阳性克隆scFv基因,亚克隆至表达载体pPICZαC,获得重组酵母表达质粒pPICZαC-scFv,线性化pPICZαC-scFv并高效电转P.pastoris（X-33）,对转化子进行抗性梯度筛选得到一株高效表达的X-33-Pp-SMW-12-6菌株。对获得的菌株先后进行表达条件的优化、优化条件下的诱导表达及单链抗体性质研究。结果表明：P.pastoris-scFv的质量接近其亲本E.coli-scFv的质量,X-33-Pp-SMW12-6在优化条件下的表达产量达28mg/L,比未优化前的产量提高了约8mg/L,经纯化后抗体纯度可达85%以上。因此,利用P.pastoris表达系统制备抗速灭威单链抗体比细菌表达系统更有效、更经济。     

猫血清白蛋白在毕赤酵母中的分泌表达

【目的】 在毕赤酵母中构建猫血清白蛋白（feline serum albumin,FSA）表达系统,并探索其最适的基本表达条件,为FSA的生产提供一种新方法。【方法】 在GenBank上获取FSA碱基序列,进行密码子优化并合成,将优化的密码子构建到载体pPIC9K上,经PCR和双酶切验证后通过电击转化将重组质粒FSA-pPIC9K转化毕赤酵母GS115,依次经MD平板和G418筛选后进行菌落PCR获得阳性菌株,随机选取阳性菌株经甲醇诱导表达96 h后,取表达上清液进行Western blotting验证。选取表达量较高的菌株分别在不同温度（24、26、28和30 ℃）、pH （4.0、5.0、6.0、7.0和8.0）和甲醇诱导量（其中1组为每24 h添加0.5%,其余4组为每12 h分别添加0.5%、1.0%、1.5%和2.0%）的条件下诱导表达96 h,取表达上清液进行Western blotting验证。【结果】 菌落PCR结果显示,获得2条大小分别约为2.3和2.2 kb的目的基因条带和毕赤酵母AOX1基因扩增条带,诱导表达96 h后取表达上清液进行Western blotting验证,在70 ku左右出现特异性条带。通过基本条件优化发现该蛋白在28 ℃,pH为6.0且每12 h添加0.5%的甲醇条件下表达量较高。【结论】 毕赤酵母GS115可稳定表达FSA。     

低pH值处理对固定化细胞乙醇发酵的影响

利用海藻酸锰凝胶包埋，将低浓度的树干毕赤酵母(Pichia stipltis)细胞固定，其凝胶颗粒直径约2-3mm，经增殖培养，进行乙醇发酵低pH值处理研究。结果表明，以低pH值(3．8、2．8、2．4)无菌水处理海藻酸锰固定化酵母细胞凝胶珠2-3h，再恢复正常pH值5．0。当用pH值2．8无菌水处理时，细菌基本上被杀死，而酵母细胞功能不受影响；当pH值2．4时，对酵母细胞影响较大，电子显微镜下观察，酵母细胞由椭圆形变成长形或不规则形状，其乙醇代谢功能严重受损。     

细菌素Bacteriocin E50-52(H)基因设计 及其毕赤酵母表达载体构建

设计及合成细菌素Bacteriocin E50-52(H)基因,克隆到组成型分泌表达质粒pGAPZαA中构建重组质粒pGAPZα-Bacteriocin E(H),经PCR、测序验证正确后,电转化整合到毕赤酵母基因组,基因组PCR、测序验证结果表明成功构建了细菌素组成型重组表达载体,为在毕赤酵母中表达奠定了基础。     

里氏木霉木聚糖酶XYNⅡ基因在毕赤酵母中的分泌表达

采用RT-PCR方法克隆到里氏木霉Rut C-30木聚糖酶(XYN Ⅱ)的cDNA序列.测序结果表明,XYN Ⅱ的cDNA基因开放阅读框长度为669bp,编码223个氨基酸,N端1～19个氨基酸为潜在的信号肤序列,删去潜在信号肽序列,将里氏木霉木聚糖酶的基因(xyn Ⅱ)构建到巴斯德毕赤酵母分泌表达载体pPIC9K上,线性化后电击转化到巴斯德毕赤酵母中,经G418筛选和PCR鉴定后的转化子用1%的甲醇进行诱导,对重组木聚糖酶活检测显示该基因能在毕赤酵母中表达有生物活性的XYNⅡ并分泌到胞外.发酵液中的酶活在诱导培养60h达到1.45IU/mL,最适酶解温度为50℃,最适pH值为6.0.     

Methylotrophic yeast,lactic acid bacteria and glycerine as additives for sugarcane silage

The ensiling of sugarcane results in high dry‐matter (DM) loss, but the addition of glycerine may compensate for the loss during ensiling. Methanol is the most undesirable contaminant of crude glycerine destined for animal feeding. The aim of this study was to evaluate the ability of the yeast strain Pichia methanolica NCYC 1381 to reduce the methanol concentration in sugarcane silage inoculated with Lactobacillus hilgardii CCMA 0170 + glycerine. A randomized design consisted of four dose rates of glycerine (0, 4, 8 and 12% of fresh forage), three periods of silage fermentation (11, 34 and 68 d) and three combinations of microbial additives [L. hilgardii (LH), L. hilgardii plus P. methanolica (LH + PM) and without microbial additive (WI)]. The DM of the fresh sugarcane was 275 g kg^?1. The linear reduction in neutral detergent fibre caused by glycerine inclusion was probably due to a dilution effect. The LH treatment increased the concentrations of the succinic, acetic and propionic acids, and 1,2‐propanediol, and reduced the yeast population. The LH + PM treatment increased DM loss of sugarcane silage with 12% glycerine and L. hilgardii CCMA 0170 (6·1 log cfu g^?1 of FM) reduced the DM loss when compared to the silage without additives. Under the conditions of the experiment, the P. methanolica treatment did not reduce the methanol concentration in silage.     

凝集素样氧化型低密度脂蛋白受体-1的cDNA克隆及在毕赤酵母中的表达

凝集素样氧化型低密度脂蛋白受体-1(LOX-1)是氧化型低密度脂蛋白(ox-LDL)的主要受体，它与动脉粥样硬化过程密切相关。通过RT-PCR从THP-1细胞系获得LOX-1cDNA并被克隆到载体pPIC9K的α信号肽的下游。线性化的重组质粒转化Pichia pastoris GS115。阳性转化子经过BMMC液体培养基培养，上清液经聚丙烯酰胺凝胶电泳(SDS-PAGE)分析，出现1条在受体菌对照中不曾有的大小约43kDa的蛋白带。重组蛋白的功能研究在进行中。