超声破碎重组大肠杆菌释放包含体的过程研究

重组人白细胞介素 (rhIL - 6 )基因表达产物在大肠杆菌的细胞浆中以包含体的形式存在。用超声破碎法提取rhIL - 6包含体系统研究了该过程中的物理和化学因素 ,包括破碎时间 ,输出功率 ,菌体浓度 ,缓冲液pH值 ,菌体冻融及悬浮液体系等。得到了破碎提取包含体的最适条件为 :超声功率 4 80W ,菌体浓度为 1 0 0g L左右。在去离子水中经过 1 5min超声破碎 ,表达量为 2 0 %的菌体可获得包含体的含量为 4 0 %(质量分数 ,下同 )     

重组人成骨蛋白-1复性条件的优化

目的优化重组人成骨蛋白-1(rhOP-1)包涵体蛋白的复性条件。方法将表达rhOP-1的大肠杆菌菌体在冰浴下超声裂解,分离提取包涵体,用8mol/L尿素溶解,纯化后进行梯度透析复性。利用TotalLab软件分析目的蛋白二聚体的含量,用体内法和体外法测定其生物学活性。结果经纯化后,目的蛋白纯度达97%以上。最佳复性条件为4℃,pH9.0,蛋白浓度为0.4～0.8mg/ml,尿素浓度为2mol/L,L-Arg浓度为0.4mol/L;目的蛋白复性率达75%以上,复性后蛋白具有较高的生物学活性。结论已确定了rhOP-1包涵体梯度透析复性的最佳条件。     

反相高效液相色谱法测定苯扎贝特的含量

研究了采用反相高效液相色谱测定苯扎贝特含量的方法 ,以磷酸盐缓冲液 (0 0 1mol/L的磷酸二氢钾用磷酸调节pH至 3 8) 甲醇 (4∶6 )为流动相 ,采用HypersilC18柱 (5 μm ,4 6mm× 15 0mm) ,检测波长为 2 30nm ,苯扎贝特在 0 0 114～0 114mg/mL范围内线性相关 ,回归方程为 :A =1 0 5× 10 4c(mg/mL) +2 70× 10 2 ,r =0 9997,此法简便、快速、结果准确可靠     

牛对氧磷酶-1蛋白多克隆抗体的制备及其双抗夹心ELISA检测方法的建立

目的制备牛对氧磷酶-1(paraoxonase-1,PON-1)蛋白多克隆抗体,并建立PON-1的双抗夹心ELISA检测方法。方法将重组牛PON-1蛋白免疫家兔,制备多克隆抗体,经AKTA蛋白纯化系统纯化抗体。以获得的多克隆抗体为捕获抗体,牛PON-1单克隆抗体为检测抗体,HRP标记的山羊抗小鼠IgG为二抗,构建牛PON-1蛋白的双抗夹心ELISA检测法,并对多克隆抗体(1∶100、1∶200、1∶400、1∶800、1∶1 600、1∶3 200、1∶6 400、1∶12 800)及单克隆抗体浓度(0. 2、0. 4、0. 8、1. 6、3. 2、6. 4 mg/mL)、包被条件(37℃孵育2 h、4℃孵育过夜)、封闭液(1%脱脂奶粉、5%脱脂奶粉、0. 5 mol/L氯化铵)、封闭条件(37℃2 h、37℃4 h、4℃过夜)进行优化,同时验证方法的线性及准确度。采用该方法及市售试剂盒同时检测酮病组及对照组奶牛血清中的PON-1含量。结果建立的双抗夹心ELISA法最适检测条件为:单克隆抗体浓度为3. 2 mg/mL,多克隆抗体工作浓度为1∶800稀释,封闭液为5%脱脂奶粉,包被及封闭条件均为4℃过夜。牛PON-1蛋白标准品浓度在90～3 125 ng/mL浓度范围内,与A_(450)值呈良好的线性关系,标准曲线方程为y=0. 618 6 x-0. 753 6,R~2=0. 981 3;5份样品检测结果均值为764. 21 ng/mL,回收率为97. 97%。市售试剂盒和本实验建立方法检测酮病组奶牛体内PON-1含量均显著低于对照组(P 0. 001)。结论成功获得了抗牛PON-1蛋白的多克隆抗体,并建立了牛PON-1蛋白的双抗夹心ELISA检测方法,且该方法具有良好的线性及准确度。     

一种新型球形纤维素吸附剂的研究

棉花经碱化、老化和磺化得黏胶液 ,再用热溶胶转相法制得球形纤维素珠体。对纤维素珠体进行交联、接枝 ,研制出球形羧基纤维素吸附剂。在黏胶的制备过程中 ,棉与碱的最佳质量比为 1∶1,老化时间 6 0h ,磺化温度 2 3℃、磺化时间 3 0h ,CS2 用量为碱纤维素中纤维素质量的35 6 %。在成球过程中 ,V (黏胶 )∶V(变压器油 ) =(1 0∶3 5 )～ (1 0∶2 0 )。交联的最佳条件为 :交联温度 75℃ ,环氧氯丙烷用量 14mL ,碱液质量分数为 10 %。接枝的主要因素包括丙烯腈浓度 2 95mol/L、Ce4 + 盐浓度 0 0 0 72mol/L、接枝反应时间 1 0h以及接枝反应温度 2 5℃。这种球形纤维素吸附剂在不同溶液 /溶剂中的膨胀系数为 1 0 0～ 1 36 ,能经受 1 2mol/L的HCl和 1 0mol/L的NaOH水溶液 15次的反复处理 ,而且对Cr3 + 、Al3 + 、Cu2 + 、Zn2 + 金属离子的吸附容量分别为 2 8 1、14 6、49 2和 37 3mg/g。     

左旋麻黄素半生物合成研究Ⅲ.L-苯基乙酰基甲醇的提取技术

对固相生物合成的L PAC(左旋麻黄素前体 )提取技术进行了研究。结果表明 ,采用H型大孔吸附树脂从生物合成的反应产物中提取L PAC最佳。当样品液pH为 6左右、过柱流速为0 2～ 0 3BV/min、柱直径与树脂高度比为 1 5∶11,用质量分数为 70 %丙酮水溶液洗脱 ,该大孔吸附树脂对L PAC的动态吸附量为 6 1 6 9mg/g。洗脱的丙酮水溶液经减压浓缩 ,获油状L PAC粗品 ,其质量浓度不低于 0 2 4g/mL     

生物化学法处理林可霉素生产废水的工艺研究

利用内循环厌氧反应器(IC)废水处理装置,研究了有效微生物(EM)和高分子絮凝剂对林可霉素生产废水处理工艺参数,试验表明,单独使用EM时最佳的接种量为0.3%～0.4%,pH为6,处理时间为36h,处理温度取自然温度(室温),经正交实验最佳组合为B3A3C1,即最佳温度为30 0℃,最佳接种量为0.5%,最佳pH为5.处理后COD从30000 mg/L降至1000 mg/L左右,经高分子絮凝剂处理最佳的pH为8,万分之一的絮凝剂加量为(0.75～1.0)mL/250mL废液,沉降时间为1.5～2.Oh,主要污染指标COD降低到300mg/L以下,达到了国家排放标准.     

23F型肺炎链球菌多糖蛋白结合物游离蛋白含量检测方法的建立及验证

目的建立一种检测23F型肺炎链球菌多糖结合物游离蛋白含量的方法,并进行方法学验证。方法采用体积排阻高效液相色谱(size exclusion chromatography and high performance liquid chromatography,SEC-HPLC)法检测23F型肺炎链球菌多糖结合物游离蛋白含量。色谱柱:TSKgel G3000(300 mm×78 mm,5μm);流动相:10 mmol/L PBS(150 mmol/L NaCl,pH 6. 8);检测波长:214 nm;流速:0. 5 m L/min;柱温:25℃;自动进样:100μL。同时对该方法进行线性、精密度、准确度、耐用性及专属性验证。结果建立的方法在纯化载体蛋白破伤风类毒素(tetanus toxoid,TT)含量为3～18μg/mL时,与峰面积呈良好的线性关系,线性相关系数(R2) 0. 99;23F-2017001、23F-2017002、23F-2017003批供试品重复性相对标准偏差(RSD)分别为0. 1%、0. 3%、1. 0%,中间精密性RSD分别为3. 7%、8. 5%、4. 8%;加入6、8、10μg/mL纯化载体蛋白TT样品的3次检测结果回收率在93. 2%～102. 6%之间,回收率的RSD均为0. 6%;进样温度为20～30℃条件下的保留时间和峰面积相对偏倚在-0. 1%～0. 5%之间;流动相溶液中含甘氨酸及低浓度硼酸时对检测结果无干扰,硼酸浓度为20 mmol/L以上时对游离蛋白峰检测有一定干扰。结论本实验建立的方法具有良好的线性、精密性、准确性、耐用性及专属性,可用于23F型肺炎链球菌多糖结合物游离蛋白含量检测。     

重组蛇毒纤溶酶Fibrolase的复性

将Fibrolase基因克隆入表达载体pET25b(+),在大肠杆菌BL21(DE3)中进行了高效表达,目的蛋白以包涵体形式存在,占菌体总蛋白的40%左右. 比较了直接稀释法、流加稀释法和透析法的复性效率,同时研究了温度、pH 值、初始蛋白浓度、复性液中盐酸胍浓度和氧化/还原环境等对复性的影响, 得到了较为适宜的复性条件. 结果表明,采用流加稀释复性,并在4℃, pH 8.5, 1.5 mol/L盐酸胍, 1 mmo1/L GSSG, 2 mmo1/L GSH, 初始蛋白浓度10 mg/mL及稀释体积为50倍时,复性效率最高. 重组Fibrolase蛋白在复性和纯化后的收率可达25%左右.     

手性动态固定相HPLC法拆分2-甲胺基-2-邻氯苯基环己酮对映体

在Nova PakPhenyl色谱柱上建立了拆分2 甲胺基 2 邻氯苯基环己酮(氯胺酮)对映体的HPLC方法。以甲醇、c(H3PO4)=0 2mmol/L磷酸水溶液为流动相,V(甲醇)∶V(磷酸)=45∶55,再添加N 苄氧羰基 S 苯基 L 半胱胺酸(BPC)为手性动态固定相试剂,c(BPC)=6 5mmol/L,流动相流速1 0mL/min,检测波长254nm,氯胺酮对映体的分离选择性系数1 08,分离度1 94,质量浓度测定的线性范围0 1～2 0mg/mL,相对标准偏差0 30%～0 68%,最小检测限为0 78μg。     

重组HIV-1跨膜蛋白Gp41包涵体纯化条件的优化

目的优化重组HIV-1跨膜蛋白Gp41包涵体的纯化条件。方法将表达Gp41的大肠杆菌菌体高压匀浆破碎、洗涤分离提取包涵体,以不同pH值的低浓度变性剂溶解包涵体,稀释复性并用离子交换层析纯化目的蛋白,纯化产物经West-ernblot进行鉴定。结果以50mmol/LTris buffer、2mol/L,pH11.5尿素溶解的包涵体蛋白得到很好的溶解,目的蛋白复性得率大于40%。经纯化后,目的蛋白的纯度大于90%,且具有良好的反应原性。结论优化了重组HIV-1跨膜蛋白Gp41包涵体的纯化条件,为HIV-1Gp41抗原纯化工艺的放大提供了依据。     

重组人EC-SOD包涵体的稀释复性及重折叠后蛋白的纯化

目的确定重组人ECSOD包涵体体外稀释复性和复性后蛋白纯化的适宜方法。方法通过对pH、温度、包涵体蛋白浓度、尿素和精氨酸浓度及氧化还原对比率的定性定量分析,研究重组人ECSOD包涵体体外稀释复性的基本条件。采用金属离子亲和柱(IMAC)和肝素亲和柱(Heparinsepharose)对复性蛋白进行纯化,比较纯化效率的差异。结果最佳复性条件为pH8.5,尿素浓度为1.5molL,精氨酸浓度大于0.3molL,GSH∶GSSG=4∶1;复性温度及包涵体蛋白终浓度越高,复性效率越低。IMAC和Heparinsepharose亲和柱均可用来纯化复性蛋白溶液,但Heparinsepharose纯化效率较高。结论确定了重组人ECSOD包涵体体外稀释复性的最佳条件及复性后蛋白纯化的方法。     

人LNα4链LG3-4组件的优化表达及活性检测

目的优化人层粘连蛋白α4链LG3-4组件基因在大肠杆菌中的表达条件。方法分别从诱导温度、诱导时间、IPTG浓度等方面进行优化。表达产物经Ni-NTA介质纯化,MTT法检测目的蛋白对人肺癌A549细胞粘附及增殖功能的影响。结果在20℃下,1mmol/L的IPTG诱导6h,目的蛋白呈最佳可溶性表达;在37℃下,2mmol/L的IPTG诱导4h,呈最佳包涵体形式表达。纯化后目的蛋白纯度达96%,且具有良好的增强人肺癌A549细胞伸展和粘附的功能。结论已确立了hLNα4LG3-4重组蛋白在大肠杆菌中的最佳表达条件。     

Liquid-Liquid Extraction of Low Molecular-Weight Proteins by Selective Solubilization in Reversed Micelles

Abstract

The effects of pH and salt concentration on the solubilization of ribonuclease-a, cytochrome-c and lysozyme in Aerosol OT/isooctane reversed micelle solutions have been studied to explore the potential for employing this solvent system in the large-scale recovery and concentration of proteins using liquid extraction. For pH values below the isoelectric point, pI, of the protein, solubilization was high, probably owing to strong electrostatic-interactions between the positively charged proteins and the anionic surfactant heads forming the inner micelle wall. Above the pI, the proteins could not be solubilized, probably because of unfavorable electrostatic repulsions between the like-charged proteins and surfactants. At low ionic strength and neutral pH, complete solubilization of the proteins was observed. As the ionic strength was increased, there was an abrupt decrease in solubilizing power of the reversed micelle solutions; the salt concentration at which this occurred was different for each protein. A mixture of the three proteins was cleanly resolved using a single extraction step and two stripping steps. The conditions in each step were selected according to the results of the single protein extraction studies.     

A Roast-Leach Process for Extraction of Rare Earths from Complex Monazite-Xenotime Concentrates

Abstract

The proposed process approaches the problem of solubilizing rare-earth phosphates (monazite and xenotime) found at the Pea Ridge iron mine in Sullivan, MO, from both a pyrometallurgical and hydrometallurgical point of view. It utilizes a roasting operation that converts the rare-earth phosphates to rare-earth oxides (REOs), which eliminates some costly and hazardous processing steps currently in practice. Different combinations of roasting temperatures and acid concentrations have been investigated to selectively extract the rare-earth values. Cerium is selectively solubilized by roasting at 427[ddot]C and leaching with a sulfuric acid concentration of 265 g/L. After the cerium is removed, the neodymium and lanthanum can be solubilized at a roasting temperature of 500[ddot]C and a sulfuric acid concentration of 88 g/L. Finally, neodymium, praseodymium, and yttrium are solubilized at a roasting temperature of 871[ddot]C and a sulfuric acid concentration of 265 g/L. Alternative leaching media, such as thiourea, sulfuric acid-doped thiourea mixtures, ammonium thiosulfate, nitric acid, and hydrochloric acid have also been investigated along with the addition of ultrasonic agitation. Using ultrasonics in addition to mechanical agitation, hydrochloric acid proved to be the best leaching medium. The best roasting temperatures for selective solubilization remained the same, but all of the leaching steps were conducted at 64 g/L hydrochloric acid.     

Purification and renaturation of recombinant human lymphotoxin (tumour necrosis factor beta) expressed in Escherichia coli as inclusion bodies

High level expression of recombinant human tumour necrosis factor β (rh TNF-β) in Escherichia coli results in the formation of two portions of protein, namely soluble active protein and insoluble protein which is inactive and aggregates in the form of inclusion bodies (IBs). In this study, a procedure for purification and renaturation of rh TNF-β from inclusion bodies has been designed and verified experimentally with a product purity of more than 90% and a recovery of about 30%. The procedure includes washing of IBs with specific wash buffer (Triton X-100/EDTA/lysozyme/PMSF), their solubilization with 8 mol dm^?3 alkaline urea, purification with ion-exchange columns, refolding with renaturation buffer and finally concentration and desalination with an ultrafiltration membrane. The characteristics of the renatured protein were identical with those of purified protein from the soluble fraction as demonstrated by (1) SDS-PAGE, (2) cytotoxic activity on mouse L929 cells, (3) N-terminal amino acid sequence, and (4) gel filtration chromatography.     

Solubilization of fatty soils by a radiotracer technique

A technique for measurement of solubilized radiotagged triolein and tristearin fatty soils is described. By using surfactant solutions under standardized conditions of temperature and agitation, the solubilized soils are removed from emulsified materials by filtration through 0.1 and 0.01 micron-pore size of filters. The radiotagged fat is recovered by solvent extraction from the clear filtrate by salting-out under centrifugal force and is measured by conventional counting technique. The nonionic alkanol- and alkylphenol-ethylene oxide (EO) adducts solubilized up to 0.058% triolein (weight percentage at 75°C.) while anionic surfactant and sodium tripolyphosphate solubilization was negligible. These findings suggest for these nonionics that solubilization is one of the main, if not the controlling factor in the mechanism of soil removal. Nonionic solubilization was at a maximum for 10 molar EO adducts and at near cloud-point temperatures. For the same surfactant more triolein than tristearin was solubilized, possibly on account of spatial considerations. For tridecanol-10 EO at 0.25% the heat of solubilization of triolein, ΔH_s, was 15 kcal/mole while the heat of micellization of the adduct was 1.3 kcal/mole of adduct. Differences in the colloidal ion lengths of the micelles and their aggregation numbers may explain the differences in solubilization between the anionic and nonionic surfactants tested.