高产L-5-甲基四氢叶酸基因工程菌的构建与表达

L-5-甲基四氢叶酸是叶酸最具生物活性的形式,也是唯一可以穿透血脑屏障的叶酸类药物,有极大的应用价值。为提高菌体内L-5-甲基四氢叶酸的含量,将5,10-亚甲基四氢叶酸还原酶基因metF插入载体质粒,并将重组质粒pET-22b-metF转化至E.coliBL21(DE3),乳糖诱导蛋白表达。成功构建的重组菌经诱导后,L-5-甲基四氢叶酸量比原始菌株中提高28%,达到66μg/g菌体。本研究成功的为生物合成L-5-甲基四氢叶酸寻找到一种新的途径。     

基因工程法生产L-5-甲基四氢叶酸的研究

目的在E.coli BL21(DE3)中表达5,10-亚甲基四氢叶酸还原酶(MTHFR),并检测MTHFR的表达情况及L-5-甲基四氢叶酸的增加量。方法采用PCR法获得MTHFR基因(metF)全长DNA,分别在其5'端和3'端加上BamHⅠ和SacⅠ酶切位点。经BamHⅠ和SacⅠ双酶切后,连接到经同样双酶切的表达载体pET-28a(+)中,经限制性酶切、菌落PCR和测序验证正确后,转化至E.coli BL21(DE3),经IPTG诱导重组蛋白表达,SDS-PAGE检测MTHFR表达量,HPLC检测L-5-甲基四氢叶酸量。结果经酶切与测序鉴定证实原核重组载体pET28a-MTHFR构建成功,表达产物相对分子质量约为33 000,与MTHFR大小相符,工程菌的L-5-甲基四氢叶酸的产量显著增加。结论通过将MTHFR的基因导入L-5-甲基四氢叶酸产生菌能够提高L-5-甲基四氢叶酸产量。     

菌体中L-5-甲基四氢叶酸的HPLC法检测

目的:建立高效液相色谱法测定菌体中L-5-甲基四氢叶酸(L-5-MTHF)的方法。方法:加热法破碎细胞,强碱性阴离子交换树脂纯化,高效液相色谱柱为C18反向柱,流动相为K2HPO4(0.02 mol.L-1,pH7.2)-甲醇(84∶16)溶液,流速为1.0 mL.min-1,柱温为40℃,检测波长为290 nm。结果:L-5-MTHF在0.5～50μg.mL-1范围内线性良好,r=0.999 5,RSD为1.11%。结论:色谱法灵敏、准确、重现性好,可用于菌体中L-5-甲基四氢叶酸的定性与定量。     

亚甲基四氢叶酸还原酶的纯化及催化性质研究

对亚甲基四氢叶酸还原酶(methylenetetrahydrofolate reductase,MTHFR)的体外酶促反应性质进行研究。通过盐析、DEAE-Sepharose fast flow柱层析纯化猪肝中的MTHFR,采用荧光法分析测定该酶纯化结果和酶促动力学性质。结果表明,MTHFR的纯化倍数可达20倍以上;测得最适酶促反应条件为pH 6.8,37℃;NADPH浓度1.2 mmol/L;以5,10-亚甲基四氢叶酸为底物测得米氏常数km=70.3μmol/L。研究发现,别构抑制剂S-腺苷甲硫氨酸(AdoMet)可使MTHFR的体外活性降到原活性的60%以下。本文初步探讨了MTHFR的催化性质,对MTHFR催化活性的主要影响因素进行研究,为酶促法合成5-甲基四氢叶酸的进一步研究奠定了基础。     

5,10-亚甲基四氢叶酸还原酶基因的研究进展

叶酸是一种水溶性B族维生素,其主要生理功能是在生化反应中转移一碳单位即某些氨基酸在分解过程中产生的含有一个碳原子的基团,如甲基、亚甲基、次甲基等,为核苷酸单位从头合成DNA和RNA提供一碳单位.     

亚甲基四氢叶酸还原酶基因多态性与个体化叶酸补充概述

亚甲基四氢叶酸还原酶（MTHFR）是机体叶酸代谢过程中最为关键酶,参与机体许多生理生化过程。科学研究证实MTHFER基因多态性引起的酶活性降低可导致叶酸代谢障碍,该遗传特征与许多疾病易感性,临床药物治疗作用及不良反应相关,而适当的补充叶酸能够在一定程度上可规避这种遗传因素所致疾病,本文就目前MTHFR基因多态性与疾病关系作一综述,并就结合基因型制定个体化叶酸补充做一预测和评估。     

亚甲基四氢叶酸还原酶C667T基因多态性与晚期胃癌患者对5-FU化疗疗效的相关性

目的 观察亚甲基四氢叶酸还原酶(MTHFR)C667T基因多态性与5-氟尿嘧啶(5-FU)为基础的化疗方案治疗晚期胃癌的疗效间的关系.方法 收集经病理学确诊的晚期胃癌59例.所有病例化疗前抽取外周静脉血,提取DNA,用连接酶检测反应技术(LDR)检测研究对象的MTHFR基因型.所有患者经5-FU为基础的联合化疗方案化疗.结果 59例晚期胃癌患者中,21例(35.59%)为MTHFR C/C基因型,22例(37.29%)为MTHFR C/T基因型,16例(27.12%)为MTHFR T/T基因型.其中,4例CR,14例PR,19例SD,22例PD,总有效率为30.51%(18/59).MTHFRT/T基因型患者的化疗有效率(68.75%)明显高于C/T基因型患者(18.18%)(P＜0.01),也明显高于C/C基因型(14.29%)(P＜0.01).结论 MTHFR基因型对预测以5-FU为基础化疗方案治疗晚期胃癌的疗效具有较好的临床意义.     

L-5-甲基四氢叶酸钙在SD大鼠体内的药代动力学研究

目的建立测定SD大鼠血浆中L-5-甲基四氢叶酸钙(5-MTHF)浓度的高效液相色谱-串联质谱法(HPLC-MS/MS法),初步考察5-MTHF在SD大鼠体内的药代动力学(PK)参数。方法给予6只雄性SD大鼠口服1.5×10^-2mmol·L^-1·kg^-15-MTHF,采集血样用于HPLC-MS/MS分析。用蛋白沉淀法处理血浆样本,色谱柱:Grace Altima HP C₁₈,流动相A:甲醇-乙腈-异丙醇-乙醇-水-甲酸-二甲基亚砜(25∶25∶25∶25∶0.1∶1);流动相B:甲醇-水-甲酸(10∶90∶0.1);梯度洗脱:0.40 mL·min^-1。考察该方法的线性范围、准确度和精密度、回收率、基质效应及稳定性。用WinNonlin软件计算5-MTHF在SD大鼠体内的PK参数。结果 5-MTHF在10.00~1.00×10~4ng·mL^-1内线性关系良好,回归方程为y=1.01×10^-3x+1.82×10^-3(r=0.998 3),日内和日间的准确性和精密度、回收率、基质效应及稳定性符合要求。5-MTHF在SD大鼠体内的PK参数:不扣除本底5-MTHF浓度值的t_1/2为(15.23±5.76)h,AUC_0~t为(2788.52±348.86)ng·mL^-1·h,扣除本底5-MTHF浓度值的t_1/2为(1.57±0.34)h,AUC_0~t平均值为(971.80±237.58)ng·mL^-1·h。结论本研究建立了一种灵敏的、准确的用于SD大鼠血浆中5-MTHF浓度测定的HPLC-MS/MS法。     

妊娠期高血压疾病与亚甲基四氢叶酸还原酶基因的相关性研究进展

妊娠期高血压疾病是妊娠晚期出现的特发性高血压综合征,是导致孕产妇和围产儿发病率和死亡率升高的重要原因,其发病率高达7%～10%,其病因尚无定论.妊娠期高血压疾病的病因可能为内皮细胞的损伤或功能紊乱,随着后基因时代的到来,人们研究发现妊娠期高血压疾病与遗传因素也有重要的关系.近年来有研究显示亚甲基四氢叶酸还原酶活性的变化可能导致血高同型半胱氨酸,从而引起内皮细胞损伤或内皮功能的紊乱,由此推测亚甲基四氢叶酸还原酶基因可能与妊娠期高血压疾病的发生有关.本文章就近年来亚甲基四氢叶酸还原酶基因与妊娠期高血压疾病的关系的研究进展作一综述.     

长治市女性亚甲基四氢叶酸还原酶基因多态性分析

目的了解亚甲基四氢叶酸还原酶(MTHFR)C677T基因多态性在长治女性人群的分布情况。方法采用实时荧光定量聚合酶链反应(q PCR)方法,对829例女性的MTHFR C677T基因多态性进行检测。结果本地区女性人群MTHFR 677位点CC、CT、TT基因型频率分别为17.61%,45.11%,37.27%;C和T等位基因频率为40.17%和59.83%。本地区女性MTHFR的基因型和等位基因频率与郑州、廊坊地区比较差异无统计学意义(P>0.05),与珠海、云南、温州、苏州、西安、长春、齐齐哈尔女性比较差异有统计学意义(P<0.05)。结论本研究中MTHFR C677T基因多态性在女性人群中的分布符合Hardy-Weinberg平衡,为本地女性优生优育及叶酸代谢相关疾病提供流行病学资料。     

重组人SOD工程菌发酵表达条件的优化

采用正交试验优化重组人超氧化物歧化酶工程菌的LB培养基,得到培养基最佳配比(g/L):蛋白胨12,酵母膏5,葡萄糖1和氯化钠10.以此为基础优化基因工程菌发酵条件,确定培养基初始pH 6.5～7.5,装液量20％,菌体密度(D600) 0.55～1.4,42℃诱导表达2～3h,目的蛋白相对表达量由优化前的25％提高至4...     

基因工程菌发酵表达核苷磷酸化酶条件的优化

目的 优化苷磷酸化酶（包括嘌呤和嘧啶核苷磷酸化酶）基因工程菌的发酵表达条件。方法通过工程菌摇瓶培养,测定吸光度D值,考马斯亮蓝（Bradford）法测定蛋白,SDS—PAGE电泳和凝胶成像扫描分析表达量,优化表达条件;通过正交试验优化50L发酵罐发酵条件。结果摇瓶培养起始pH为7．0～7．2,于30℃培养4h,加入终浓度为0．4mmol／L的异丙基硫代半乳糖苷（IPTG）诱导8h后收获菌体,可得到较高的生物量和重组酶蛋白表达量。50L发酵罐的最佳条件为起始pH为7．0～7．2,于32oC培养4h,加入终浓度为0．4mm01／L的IPTG诱导9h后收获菌体,每升发酵液可得2g以上的酶蛋白。结论基因工程菌发酵表达核苷磷酸化酶产量较高,可工业化生产．为酶法合成核苷酸类似物的研究奠定了基础。     

Bioavailability and Related Pharmacokinetics in Man of Orally Administered L-5-Hydroxytryptophan in Steady State

Abstract The bioavailability of orally administered L-5-hydroxytryptophan in steady state was investigated at four increasing multiple dose levels in five patients suffering from various myoclonic disorders. An L-aromatic amino acid decarboxylase inhibitor was co-administered in all the experiments. The disposition pharmacokinetics of the amino acid had been established in the same patients in preceding intravenous single dose experiments. The finding of a direct proportionality between the size of the oral dose level of L-5-hydroxytryptophan and the corresponding areas under the plasma concentration curves within a dosage interval at steady state strongly indicates dose independent, linear pharmacokinetics of the compound. The systemic availability of L-5-hydroxytryptophan exhibited an interindividual range of 47–84%, with a mean value of 69.2% ±4.7 S.E.M. The absorption took place at a rather slow rate as judged from times of 1.8 to 3.3 hours elapsing from administration of the compound until occurance of the maximum measured plasma concentrations. Transitory nausea and vomiting were only recognized in few instances during the gradual building up of increasing steady state levels of L-5-hydroxytryptophan in the patients, and the importance of a slow initiation of therapeutical treatment with the amino acid is emphasized.     

N-methylation of biogenic amines. I. Characterization and properties of an N-methyltransferase in rat brain using 5-methyltetrahydrofolic acid as the methyl donor

Some properties and kinetics of a recently identified brain N-methyltransferase requiring 5-methyltetrahydrofolic acid as the methyl donor are described in this paper. In addition to the assay using dopamine as substrate, the more stable substrate, 3-hydroxy-4-methoxyphenethylamine with a rapid extraction procedure was also used. The pH optimum of the enzyme, about pH 6·4, was found to differ from that previously found with dopamine in the presence of metabisulfite. The reaction products were identified by column and thin layer chromatography and indirectly by testing various N-methyl-, dimethyl- and dimethoxy-derivatives of dopamine for their ability to be N-methylated. The reaction was a linear function of time and enzyme concentration. The K_m for 5-methyltetrahydrofolic acid was 2.5 × 10^?5 M. The kinetic experiments to determine the K_m for dopamine and 3-hydroxy-4-methoxyphenethylamine showed an anomalous behaviour of the saturation curve. When the results were plotted S/v vs S, a non-linear curve was obtained suggesting that the enzyme was behaving as an allosteric protein. Although further kinetic experiments are needed to confirm these results, a possible regulatory function can be postulated for this enzyme. In. addition to dopamine and its derivatives, amines such as tryptamine, serotonin and amphetamine were also N-methylated under the same conditions.     

两株乳酸菌的分离及发酵性能研究

目的从酸奶中分离乳酸菌并对其发酵性能进行研究。方法选用较为简单的培养基,通过调节pH值进行乳酸菌的分离,以液体发酵研究其发酵性能。结果分离出2株乳酸菌,经鉴定为嗜热链球菌（St）与保加利亚乳杆菌（Lb）,其活性均较高。结论通过调节pH值从酸奶中可分离出2株乳酸菌;St与Lb按1∶1混合发酵时pH值下降较快（pH值最终达3.5左右）,可得到感官指标较好的酸奶。     

Serotonin synthesis with rat brain synaptosomes. Effects of L-dopa, L-3-methoxytyrosine and catecholamines

Experiments were done with a fraction of rat brain containing mitochondria and synaptosomes. L-DOPA∥ (K_t 0·3 mM) and L-3-methoxytyrosine (K_t 0·5 mM) were found to be competitive inhibitors of tryptophan accumulation, while dopamine and l-noradrenaline had no effect on the accumulation of tryptophan. Furthermore, L-DOPA (K_t 5·6 μM) was about 100 times more potent than L-3-methoxytyrosine in inhibiting the synthesis of 5HT from tryptophan. The inhibition of 5HT synthesis by L-DOPA appeared to be competitive to tryptophan and was not linear at concentrations of L-DOPA higher than 1 μM. L-DOPA also interfered with the rate of deamination of 5HT synthesized in vitro. Dopamine and l-noradrenaline also inhibited the synthesis of 5HT.     

盐酸5-氨基酮戊酸脂质体的制备与表征

目的 研究盐酸5-氨基酮戊酸(5-ALA)脂质体处方组成及制备工艺. 方法 采用薄膜分散 pH梯度法制备5-ALA脂质体,以包封率为评价指标进行正交实验筛选出最佳处方,采用高效液相色谱(HPLC)法测定其包封率. 并对其粒径、电位等理化性质进行研究. 结果最佳处方工艺为：卵磷脂与胆固醇的质量比为6：1,卵磷脂与5-ALA质量比为6：1,孵育温度为60 ℃. 所制脂质体为乳白色,平均粒径为100 nm,Zeta电位为-40 mV,平均包封率为65.0%. 结论 该制备方法 得到的5-ALA脂质体处方合理,工艺可行,包封率较高.     

麦考酚酸产生菌液体发酵条件的优化

研究了短密青霉菌L产生麦考酚酸的发酵工艺条件，包括斜面培养基种类、初始pH、培养温度、碳氮源种类等，并通过均匀设计法优化了发酵培养基的组成。在优化条件下，摇瓶发酵单位达8565μg／ml，比优化前提高了89％。