乙肝表面抗原结合蛋白SBP在毕赤酵母中的表达纯化及功能鉴定

乙肝表面抗原结合蛋白(HBsAg binding protein,SBP)是本实验室发现的一种人源蛋白,该蛋白与人乙型肝炎病毒HBV表面抗原HBsAg存在特异性的结合能力。此前的研究证实SBP具有增强乙肝疫苗免疫效果的作用。为进一步研究该蛋白的生理功能和作用机制,利用毕赤酵母表达系统进行了SBP的表达菌株构建,筛选得到了SBP的高效表达菌株。发酵产物经过分离纯化,最终得到了大量高纯度的真核来源的目的蛋白。通过SDS-PAGE、高效液相色谱、Western blotting和质谱鉴定,证实所得到的蛋白具有较高的纯度和完整性。通过ELISA方法初步证实了其与乙肝表面抗原具有较好的结合能力。该研究为进一步进行SBP的体内外功能研究及免疫增效研究打下了基础。     

一种乙肝表面抗原结合蛋白可作为疫苗佐剂

乙肝表面抗原结合蛋白(HBsAg binding protein, SBP)是本实验室发现的一种可以与乙肝表面抗原HBsAg特异性结合的人源蛋白,该蛋白已经被证实具有增强乙肝疫苗免疫效果的作用.目前,我们已经利用毕赤酵母表达系统获得了能够分泌表达SBP的毕赤酵母表达菌株.本研究通过对上述菌株的发酵产物进行超滤和亲和层析纯化,获得了一定量的高纯度重组SBP蛋白,并利用酶联免疫吸附检测(ELISA)法和表面等离子体共振(SPF)法分别对重组SBP进行了体内外生物学活性的初步检测,证实其具有与HBsAg结合的能力,并求得了二者之间的亲和常数.将重组SBP作为乙肝疫苗增效剂与乙肝疫苗共同免疫小鼠,SBP增效组小鼠与对照组相比,血清中HBsAg抗体显著升高,表明SBP在体液免疫方面对乙肝疫苗具有显著的增效作用.上述结果表明,SBP有望作为乙肝疫苗的免疫佐剂,在乙型肝炎防治方面有重要意义.     

人源性抗HBsAg抗体Fab段在酵母中的表达

通过分步整合的方式,将人源性抗乙肝表面抗原（HBsAg）抗体Fab的轻、重链基因分步整合到巴斯德毕赤（Pichia pastoris）酵母GS115菌株的染色体上,经甲醇诱导,成功地分泌表达出抗HBsAg抗体的Fab片段,表达量达50～80mg/L。ELISA结果显示重组酵母分泌表达出的Fab具有较强的结合HBsAg的能力。通过抗Fab的抗体柱亲和层析,纯化出了纯度较高的Fab产品。     

重组胰高血糖素样肽-1与人血清白蛋白融合蛋白的纯化及活性研究

利用构建的重组菌株Pichia pastoris GS115/GLP-1/HSA,在10L发酵罐中表达了胰高血糖素样肽-1与人血清白蛋白融合蛋白(GLP-1/HSA),表达量为63.6mg/L。发酵液经中空纤维柱浓缩、疏水层析、阴离子交换层析和凝胶过滤分离纯化,获得了较高纯度的GLP-1/HSA,经HPLC分析纯度达95.8%。进一步的体内活性分析结果表明,GLP-1/HSA不仅具有天然GLP-1的生物活性,而且在给药后4h仍能发挥显著性降血糖作用。以上结果表明,利用Pichia pastoris分泌型表达系统和建立的分离纯化方法,能获得大量较高纯度的GLP-1/HSA,为进一步研究和开发能够用于糖尿病临床治疗的长效GLP-1类似物奠定了基础。     

毕赤酵母表达的HBV全长Pres蛋白的分离纯化

巴斯德-毕赤酵母工程菌株GS115-PreS经发酵在甲醇诱导下可高效表达分泌型全长PreS蛋白。Western blot证明发酵液中存在着可溶性的分子量为48kD的PreS蛋白和蛋白质颗粒,蛋白质颗粒主要成分为48kD的全长Pres蛋白和28kD的S蛋白,电镜观察发现蛋白质颗粒直径为30nm。发酵液经过脱盐、浓缩处理后,上清液经DEAESFF阴离子交换柱得到纯化的PreS蛋白;超速离心和蔗糖密度梯度离心得到蛋白颗粒。该颗粒的主要组分为全长PreS蛋白（PreS1＋PreS2＋S）,还有少量的主蛋白（S）。ELISA检测证明全长PreS蛋白和蛋白颗粒有着良好的抗原性, P/N显示蛋白颗粒的抗原性比PreS蛋白的抗原性高。     

重组胰高血糖素样肽-1与人血清白蛋白融合蛋白的纯化及活性研究

利用构建的重组菌株Pichia pastoris GS115/GLP-1/HSA,在10L发酵罐中表达了胰高血糖素样肽-1与人血清白蛋白融合蛋白（GLP-1/HSA）,表达量为63.6mg/L。发酵液经中空纤维柱浓缩、疏水层析、阴离子交换层析和凝胶过滤分离纯化,获得了较高纯度的GLP-1/HSA,经HPLC分析纯度达95.8%。进一步的体内活性分析结果表明,GLP-1/HSA不仅具有天然GLP-1的生物活性,而且在给药后4 h仍能发挥显著性降血糖作用。以上结果表明,利用Pichia pastoris分泌型表达系统和建立的分离纯化方法,能获得大量较高纯度的GLP-1/HSA,为进一步研究和开发能够用于糖尿病临床治疗的长效GLP-1类似物奠定了基础。     

头孢菌素脱乙酰酶的重组表达及固定化

本文构建了利用trp启动子表达头孢菌素脱乙酰酶(CAH)的重组大肠杆菌DH5α-pCAH。重组菌在7L发酵罐(装液量2L)中发酵28 h,发酵液OD_(600)达到27,产酶313 kU/L发酵液,粗略估算重组蛋白占细胞总蛋白的70%。发酵生产的重组CAH粗酶液经过硫酸铵分级沉淀分离纯化和超滤除盐浓缩两步操作,纯化倍数为1.44,总酶活回收率56%,聚丙烯酰胺凝胶电泳检测纯化后蛋白没有明显杂蛋白条带出现。纯化后的CAH共价结合固定在环氧基载体LX-1000EP(c)上,通过对固定化条件的优化最终得到固定化酶比活443 U/g。该固定化酶重复催化50 mL 5%7-ACA底物100次后,酶活没有降低。     

Vero细胞狂犬病疫苗在凝胶柱层析工艺中样品浓度对纯化效果的影响

为研究浓缩Vero细胞狂犬病疫苗蛋白浓度对纯化效果的影响。用超滤浓缩法对狂犬病疫苗进行不同倍数的超滤浓缩,将浓缩后不同倍数的样品分别用凝胶柱层析法进行纯化,结果样品浓度在40mg/ml以下时,狂犬病毒收集液中杂蛋白去除率可达99%以上,小牛血清含量在25-37.5ng/ml之间;浓缩超过40mg/ml时,并随着样品浓度的增加,狂犬病毒收集液中杂蛋白去除率逐渐下降,且有病毒丢失,因此采用凝胶柱层析法纯化狂犬病疫苗,蛋白浓度应低于40mg/ml。     

海参i-型溶菌酶在毕赤酵母基因工程菌中优化表达及纯化

将实验室已构建的毕赤酵母基因工程茵(pPIC9K-SjLys/GS115)作为海参i-型溶茵酶生产菌株,本研究分别从甲醇浓度、培养基pH、温度和诱导时间对其产酶发酵条件进行优化.实验得出甲醇诱导浓度为1.0％,发酵培养基初始pH 6.0,温度30℃,培养96 h为最佳目的蛋白表达条件,其发酵液中海参i-型溶菌酶含量达10.63 mg/L.将发酵液经离心和超滤浓缩后得到上清液,再经离子交换和凝胶过滤层析纯化获得海参i-型溶菌酶产品,其酶活力达826.44 U/mg.经测定该酶对革兰氏阳性菌溶壁微球菌和革兰氏阴性菌副溶血弧菌均具有明显的抑菌作用.     

毕赤酵母发酵生产颗粒性乙肝表面抗原的条件优化

乙型肝炎病毒的流行对人们的生命健康造成了极大的威胁, 而有效准确的诊断和预防性疫苗是阻止其流行的主要手段, 乙肝表面抗原是诊断试剂和疫苗的主要成分。本试验在构建稳定表达HBsAg的毕赤酵母菌株后, 对其发酵条件进行了研究。采用摇瓶分批培养方法, 探讨了不同培养基、溶解氧、诱导物甲醇的浓度以及pH值等因素对菌体生长与重组蛋白表达的影响。在10 L发酵罐上采用分批补料培养的方法研究了进行扩大培养生产重组HBsAg。结果表明, FBS无机盐合成培养基是理想的工业发酵培养基, 溶解氧对菌体的生长与表达有显著的影响, 甲醇诱导最佳终浓度为1% (V/V), 发酵的最适pH值为5.4~6.0。发酵罐放大培养后, ELISA和 SDS-PAGE分析表明重组HBsAg获得了高效表达, 最终菌体生物量达到310 OD600, 表达量达到27 mg/L。电子显微镜观察表达重组乙肝抗原可以自组装为22 nm类病毒颗粒, 为HBV的新一代早期血清学诊断和疫苗的大规模生产提供了一定的参考。     

人源性抗HBsAg Fab抗体的发酵生产研究

为了适应工业生产的需要,利用fed—batch方法,重组人源性抗HBsAg Fab抗体酵母工程菌在30L发酵罐中进行了高密度发酵,发酵最适温度30℃,pH值范围5．0～5．3,溶氧范围20％～30％。发酵液OD600值达到300时开始诱导,甲醇最佳诱导浓度为10mL／L。重组人源性抗HBsAg Fab抗体经离子交换层析纯化,纯化产品经SDS-PAGE、Western blot进行分析和ELISA方法进行活性测定。结果显示,重组Fab抗体在Fed-batch发酵系统中可高效表达,经过192h的发酵生产,重组人源性抗HBsAg Fab抗体的表达量可达412mg／L。发酵上清经过离子交换层析纯化,获得纯度为95％的重组Fab抗体,该Fab抗体经ELISA分析具有较高的HBsAg抗原亲和力和特异性。结果证实可以通过高密度发酵毕赤酵母工程菌来高效生产重组人源性抗HBsAg Fab抗体,为后续的工业化生产应用奠定了基础。     

Bacterial fermentation of cheese whey for production of a ruminant feed supplement rich in curde protein.

A simple and efficient process for the production of a ruminant feed supplement, rich in crude protein (defined as total N X 6.25), by bacterial fermentation of cheese whey has been developed. The lactose in unpasteurized whey is fermented to lactate acid by Lactobacillus bulgaricus at a temperature of 43 degrees C and pH 5.5. The lactic acid produced is continually neutralized with ammonia to form ammonium lactate. The fermented product is concentrated by evaporation to a solids content of about 70% and adjusted to pH 6.8 with additional ammonia. The concentrated product contains about 55% crude protein. Approximately 6 to 8% of the crude protein is derived from bacterial cells. 17% from whey proteins, and 75 to 77% from ammonium lactate. The efficiency of conversion of lactose to lactic acid usually exceeds 95%. The fermentation time is greatly reduced upon the addition of 0.2% yeast extract or 0.1% corn steep liquor as a source of growth factors. Whey containing lactose at concentrations up to 7% can be fermented efficiently, but at higher concentrations lactose is fermented incompletely. The process has been scaled up to a pilot plant level, and 40 tons of concentrated product were produced fro animal feeding trials, without ever encountering putrefactive spoilage.     

Bacterial fermentation of cheese whey for production of a ruminant feed supplement rich in curde protein.

A simple and efficient process for the production of a ruminant feed supplement, rich in crude protein (defined as total N X 6.25), by bacterial fermentation of cheese whey has been developed. The lactose in unpasteurized whey is fermented to lactate acid by Lactobacillus bulgaricus at a temperature of 43 degrees C and pH 5.5. The lactic acid produced is continually neutralized with ammonia to form ammonium lactate. The fermented product is concentrated by evaporation to a solids content of about 70% and adjusted to pH 6.8 with additional ammonia. The concentrated product contains about 55% crude protein. Approximately 6 to 8% of the crude protein is derived from bacterial cells. 17% from whey proteins, and 75 to 77% from ammonium lactate. The efficiency of conversion of lactose to lactic acid usually exceeds 95%. The fermentation time is greatly reduced upon the addition of 0.2% yeast extract or 0.1% corn steep liquor as a source of growth factors. Whey containing lactose at concentrations up to 7% can be fermented efficiently, but at higher concentrations lactose is fermented incompletely. The process has been scaled up to a pilot plant level, and 40 tons of concentrated product were produced fro animal feeding trials, without ever encountering putrefactive spoilage.     

甲醇诱导条件和方式对乙肝表面抗原产率的影响

发酵条件是影响汉逊酵母表达乙肝表面抗原的重要因素,通过对甲醇诱导浓度、方式及诱导周期的调控,研究了不同发酵条件对汉逊酵母表达乙肝表面抗原的影响。结果表明：控制甲醇流加速率15ml／h、甲醇诱导周期40h有利于发酵过程中乙肝表面抗原产率的提高。在发酵末期,细胞浓度最高达410mg／ml,抗原水平达46mg/l。     

Improved brewing of millet beer – (OYOKPO) and production of single cell proteins from the spent grain

The improved method for preparing Oyokpo a Nigerian fermented beverage from millet, and the preparation of single cell proteins from the spent grain is described. Improvement of the brew was made by controlled malting, mashing and brewing with a pure culture of Saccharomyces cerevisiae. It had a reducing sugar content of 19.73 g/100 ml before fermentation and after fermentation 5.56% alcohol, 0.58 g/100 ml titratable acidity as acetic acid, a final pH of 4.2 and consisted of a yellowish clear liquid, slightly sour. The native brew had a reducing sugar content of 7.37 g/100 ml before fermentation and after fermentation, 2.40% alcohol, 0.43 g/100 ml titratable acidity, a final pH of 3.8 and consisted of a creamy yellowish liquid with a very sour taste. Fermented spent grain gave a higher protein yield compared to unfermented or ground millet. The lipids, proteins and crude fibre were 4.94%, 11.20% and 4.33% respectively for ground millet, 12,79%, 23.77% and 19.46% respectively for unfermented spent grain and 19.61%, 47.28% and 32.09% respectively for fermented spent grain. The high protein and fibre content of the fermented spent grain points to its potential as a feed supplement for ruminants.     

Hydrolysis and acidogenic fermentation of gelatin under anaerobic conditions in a laboratory scale upflow reactor

Summary Gelatin was dissolved in a mineral salts medium for growth under carbon limitation and fed to a mixed population of bacteria in a lab-scale upflow reactor for hydrolysis and acidogenic fermentation under anaerobic conditions, at pH=7 and 30°C.With the shortest applied liquid retention time (30 min), 84% of the protein was hydrolysed. The majority (85%) of the hydrolysed protein was fermented. The ammonia concentration in the reactor was about 1,400 mg N·l^-1.The fermentation products were mainly acetate, propionate, and valerate. Iso-butyrate, butyrate, and iso-valerate were formed to a limited extent. Gas production was very low and consisted of carbon dioxide and methane. The product composition was independent of the retention time applied. The sludge formed was slimy. No granules were formed, however a hold-up factor of 46 could be obtained.     

Production of salmosin, a snake venom-derived disintegrin, in recombinant Pichia pastoris using high cell density fed-batch fermentation

Salmosin, a snake venom-derived disintegrin, was successfully expressed in the methylotrophic yeast Pichia pastoris and secreted into the culture supernatant, as a 6 kDa protein. High-cell density fermentation of recombinant P. pastoris was optimized for the mass production of salmosin. In a 5 L jar fermentor, recombinant P. pastoris was fermented in growth medium containing 5% (w/v) glycerol at the controlled pH of 5.0. After culturing for 21 h, glycerol feeding medium was fed at one time into the culture broth. After 7 h (a total of 28 h), induction medium that contained methanol was increasingly added until the culture time totaled 75 h. Finally, these optimized culture conditions produced a high cell density of recombinant P. pastoris (dry cell weight of 113.38 g/L) and led to the mass production of salmosin (a total protein concentration of 369.2 mg/L). The culture supernatant containing salmosin inhibited platelet aggregation, resulting in a platelet aggregation of 9% compared to that of 94% in the control experiment, without culture supernatant. These results demonstrate that recombinant salmosin in culture supernatant from high cell density fed-batch fermentation can serve as a platelet aggregation inhibitor.     

In vitro antioxidant activity of liquor from fermented shrimp biowaste

Shrimp waste was fermented using lactic culture and the separated fermented liquor was lyophilized. In vitro antioxidant activities of the lyophilized powder were evaluated with respect scavenging of different radicals and quenching of generated singlet oxygen. The sample showed strong radical scavenging and singlet oxygen quenching in a dose dependent manner (p<0.001). The sample exhibited 40% scavenging of DPPH radical at 1.0mg/ml concentration while the ABTS radical scavenging was 95% even at 0.5mg/ml concentrations. Hydroxyl radical scavenging activity as measured by chemiluminescence technique showed 80% scavenging and peroxyl radical scavenging was 65% at 1.0mg/ml concentration. The singlet oxygen quenching ability of the powder was 68.3% at 1.0mg/ml concentration. The sample was found to contain low molecular weight proteins. The formation of peptides and amino acids during hydrolysis of shrimp waste protein during fermentation is expected to be responsible for the antioxidant activity. In addition as the product also contains carotenoids, it can be used as an ingredient in aquaculture feed formulations for beneficial effects.     

Dialysis Continuous Process for Ammonium-Lactate Fermentation of Whey: Experimental Tests

Laboratory experiments were conducted to validate theoretical predictions describing a dialysis continuous process for the fermentation of whey lactose to ammonium lactate, in which the fermentor contents are poised at a constant pH by adding ammonia solution and dialyzed through a membrane against water. Dried sweet-cheese whey was rehydrated to contain 230 mg of lactose per ml, supplemented with 8 mg of yeast extract per ml, charged into a 5-liter fermentor without sterilization, adjusted in pH (5.3) and temperature (44°C), and inoculated with Lactobacillus bulgaricus. The fermentor and dialysate circuits were connected, and steady-state conditions were established. A series of such conditions was managed nonaseptically for 94 days to study the process and to demonstrate efficiency and productivity. As time progressed, the fermentation remained homofermentative and increased in conversion efficiency, although membrane fouling necessitated dialyzer cleaning about every 4 weeks. With a retention time of 19 h, 97% of the substrate was converted into products. Relative to nondialysis continuous or batch processes for the fermentation, the dialysis continuous process enabled the use of more concentrated substrate, was more efficient in the rate of substrate conversion, and additionally produced a second effluent of less concentrated but purer ammonium lactate.