人生长激素基因在转基因鲤鱼体内的遗传

通过有性繁殖转移入生长激素基因鲤鱼(P₁),获得了转基因鲤鱼的子一代(F₁)和子二代(F₂);外源基因在转基因P1与普通鲤鱼杂交产生的F₁代和转基因F1代自交产生的F₂代鱼中的存在率分别为45．4％和66．7％,外源基因拷贝数在子代个体之间存在很大差异,从每细胞2拷贝到每细胞200拷贝不等;外源基因在转基因鱼子代中仍可表达具有生物功能的产物．人生长激素(h1GH),并能促进鱼的生长。     

处理柠檬酸发酵废水的高活性光合细菌P4菌株的分离鉴定*

光合细菌P₄菌株从柠檬酸发酵废水流经的污泥中分离得到。经鉴定P₄菌株为红假单胞菌属(Rhodopseudomonas),浑球红假单胞菌(R. sphacroides)。P₄菌株以乙酸钠为碳源生长良好,50小时进入稳定期(菌液OD_660nm=108—2.0)。将经过可溶化的柠檬酸发酵废水用P₄菌株的乙酸钠培养液进行处理,作用72小时后废水COD的去除率达85.3%。     

控制pH环境对出芽短梗霉胞外多糖合成的影响

采用添加 Ca CO₃ 和 HCl的方法研究了 p H对出芽短梗霉多糖发酵的影响规律。在 P2培养基中发酵2 4 h,该菌有一个强烈的产酸期 ,导致 p H迅速下降到 3.6左右。在此 p H环境下继续发酵 12 0 h,多糖产量仅为 5.9g/ L。如果用 MP2培养基 (P²⁺ 0 .5% Ca CO₃ )发酵 ,由于 Ca CO₃ 缓冲了发酵 p H的下降 ,在整个发酵过程中 p H值可以维持在 5.0以上 ,多     

添加芦荟汁对红葡萄酒发酵的抑菌效果

二氧化硫（SO₂）危害人体健康。为了寻找用于葡萄酒酿造的SO₂替代品,在赤霞珠葡萄酒自然发酵和接种发酵中比较了添加芦荟汁（80 g/L）与SO₂（60 mg/L）的抑菌和发酵效果。结果表明,添加芦荟汁促进接种酵母和内源酵母生长,SO₂抑制内源酵母生长,接种酵母、添加芦荟汁和添加SO₂显著抑制细菌和霉菌生长,SO₂比芦荟汁对细菌和霉菌生长的抑制作用大。添加芦荟汁和添加SO₂同样提高乙醇产量和减小酸度下降,降低葡萄酒的残糖量和褐变度。添加芦荟汁提高了挥发性酸度和高级醇产量,降低了接种发酵的葡萄酒色度和提高乙酸产量,降低了自然发酵的乙醛产量,而SO₂作用相反。添加芦荟汁具有减少SO₂用量的可能性。     

脱氮假单胞菌生产维生素B12过程中粪卟啉Ⅲ的测定及发酵调控

脱氮假单胞菌发酵生产维生素B₁₂过程中,副产物粪卟啉Ⅲ的积累对产物的代谢合成和分离提取有很大的影响。建立了发酵液中粪卟啉Ⅲ的HPLC快速测定方法,发酵液处理后直接进样测定,检测线性范围为12~275 μg/ml,重复性好。研究了不同供氧水平、二氧化碳浓度和pH值对发酵过程中维生素B₁₂和粪卟啉Ⅲ代谢合成的影响,并在120吨发酵罐中进行了发酵过程优化控制。结果表明:在发酵过程产物的合成期控制适当的供氧水平、控制二氧化碳浓度在8.6±0.8%、维持pH值在7.0±0.12能明显抑制卟啉Ⅲ的生物合成,同时使维生素B₁₂产量提高15%。     

小球藻病毒基因调控序列在大肠杆菌和真核藻中的调控活性研究

以小球藻病毒腺嘌呤甲基转移酶基因(amt)和主要外壳蛋白VP54基因的5′上游调控序列构建大肠杆菌和真核藻转化载体。以P_RP_L及CaMV35S启动子为阳性对照,研究了小球藻病毒来源的两种调控序列在E.coli和真核藻细胞中的启动活性。发现P_AMT在4种E.coli菌株中都具有极强的调控活性,启动Luc基因表达而产生的酶活性高于P_RP_L 50～400倍;P_VP54在DH5α中也具有较强的启动活性。同时PAMT在两种小球藻中启动GUS基因瞬时表达的能力也明显高于CaMV35S启动子,表明它们有可能在真核藻类遗传转化中具有很好的应用前景。     

鱼重组生长因子酵母的发酵研究

对含鲑鱼生长激素基因的甲醇酵母进行了发酵研究,比较了摇瓶发酵和自控罐发酵两种条件下酵母工程菌的生长和外源蛋白的表达,确定了自控罐发酵生产含鲑鱼生长激素酵母的工艺为含鱼生长因子酵母饲料添加剂的生长奠定了基础。     

碳酸钙促进丙酮酸发酵过程中α-酮戊二酸的形成

在多重维生素营养缺陷型菌株光滑球拟酵母CCTCC M202019发酵生产丙酮酸的摇瓶和发酵罐实验中发现,CaCO₃的添加对发酵液中α-酮戊二酸（α-KG）的积累有重要影响。在维生素浓度不变且供氧充分的前提下,延迟CaCO₃添加时间可明显抑制α-KG的产生,并提高丙酮酸与α-KG的碳摩尔比(C_PYR/C_αKG);而增加培养基中的CaCO₃浓度会导致αKG积累的增加。用不同物质调节发酵液中pH的实验证实：在丙酮酸发酵过程中, Ca²⁺对αKG的积累起主要作用,CO₃^2-起辅助作用,两者对α-KG的积累具有协同效应。维持培养基中CaCO₃浓度不变,改变培养基中硫胺素的浓度,对αKG的积累,特别是对C_PYR/C_α-KG值没有影响;而增加培养基中生物素的浓度,则导致αKG的浓度不断上升且C_PYR/C_α-KG值不断下降。当有Ca2+存在时,胞内丙酮酸羧化酶的活性最高可提高40%,而丙酮酸脱氢酶系的活性没有明显变化。结果表明,丙酮酸发酵过程中α-KG的形成是由于CaCO₃促进了丙酮酸羧化反应,其中Ca²⁺可显著提高丙酮酸羧化酶的活性,而CO₃^2-则有可能作为丙酮酸羧化反应的底物。     

氮源NH4Cl浓度对粪产碱杆菌发酵生产热凝胶的影响

研究了利用粪产碱杆菌 (Alcaligenesfaecalis)发酵生产热凝胶的发酵条件 ,氮源是菌体生长的限制性底物 ,单纯地提高初始底物 (氮源 )浓度并不一定能促进细菌的生长和产物的合成。在分批发酵过程中 ,底物消耗导致培养环境pH的改变也是影响细菌进一步生长和产物合成的重要因素。通过增加培养基中初始氯化铵的浓度并同时控制发酵过程的pH条件 ,得到了较高的菌体浓度 ,热凝胶的合成水平也得到了显著提高。当培养基中NH₄Cl浓度提高到3.6g/L时 ,菌体浓度达到72g/L ,热凝胶合成的产量可达 30.5g L ,比原来NH₄Cl浓度为11g L时提高了51.7%。提高菌体浓度意味着需要提高溶氧水平来满足细菌的生长和代谢。初始氮源NH₄Cl浓度的增加虽然能使菌体浓度得到提高 ,但发酵过程对溶氧的需求也相应增加 ,需要提高搅拌转速和通风以增加供氧水平。但高搅拌速率产生的高剪切力对热凝胶的凝胶性能将产生破坏作用 ,因此在发酵过程中需要综合考虑细菌培养密度对合成热凝胶产量和质量的影响。     

NH4+对L-色氨酸发酵的影响

目的:探究NH₄⁺浓度对大肠杆菌E.coli TRTH发酵生产L-色氨酸的影响。方法:通过外源添加试验,利用30 L发酵罐进行分批补料发酵试验,考察E. coli TRTH发酵生产L-色氨酸过程中生物量、L-色氨酸产量、有机酸含量、耗糖速率、发酵液中NH₄⁺浓度及质粒稳定性变化。建立了大肠杆菌合成L-色氨酸的代谢流平衡模型,应用 MATLAB 软件计算出E. coli TRTH发酵中后期代谢网络的代谢流分布。结果:发酵结果显示,利用NaOH和氨水混合补料,控制NH₄⁺浓度在120 mmol/L以下,菌体能够以较长时间和较高比生长速率保持对数生长,最终菌体生物量和L-色氨酸产量分别提高了12.16%和19.80%。随着NH₄⁺浓度的增加,发酵液中丙酮酸、乳酸及乙酸浓度均略有增加,细胞质粒稳定性下降。控制NH₄⁺浓度在120 mmol/L以下,E. coli TRTH发酵生产L-色氨酸的代谢流量分析结果表明,EMP途径的代谢流量降低7.31%,PP途径的代谢流量增加7.14%,TCA循环的代谢流量降低22.04%。结论:高浓度的NH₄⁺导致菌体生长提前结束,耗糖速率降低,产酸受阻,控制NH₄⁺浓度在120 mmol/L以下,解除了NH₄⁺对菌体生长和产物生成的抑制,使得菌体生物量和L-色氨酸产量大幅提高,实现了高密度发酵培养的目的。     

Recombinant shrimp (Litopenaeus vannamei) trypsinogen production in Pichia pastoris

Shrimp (Litopenaeus vannamei) trypsinogen has never been isolated from its natural source. To assess the production of L. vannamei trypsinogen, we engineered Pichia pastoris strains and evaluated two culture approaches with three induction culture media, to produce recombinant shrimp trypsinogen for the first time. The trypsinogen II cDNA was fused to the signal sequence of the Saccharomyces cerevisiae alpha mating factor, placed under the control of the P. pastoris AOX1 promoter, and integrated into the genome of P. pastoris host strain GS115. Using standard culture conditions for heterologous gene induction of a GS115 strain in shake flasks, recombinant shrimp trypsinogen was not detected by SDS‐PAGE and Western blot analysis. Growth kinetics revealed a toxicity of recombinant shrimp trypsinogen or its activated form over the cell host. Thus, a different culture approach was tested for the induction step, involving the use of high cell density cultures, a higher frequency of methanol feeding (every 12 h), and a buffered minimal methanol medium supplemented with sorbitol or alanine; alanine supplemented medium was found to be more efficient. After 96 h of induction with alanine supplemented medium, a 29‐kDa band from the cell‐free culture medium was clearly observed by SDS‐PAGE, and confirmed by Western blot to be shrimp trypsinogen, at a concentration of 14 μg/mL. Our results demonstrate that high density cell cultures with alanine in the induction medium allow the production of recombinant shrimp trypsinogen using the P. pastoris expression system, because of improved cell viability and greater stability of the recombinant trypsinogen. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

High-yield production of a human therapeutic protein in tobacco chloroplasts

Transgenic plants have become attractive systems for production of human therapeutic proteins because of the reduced risk of mammalian viral contaminants, the ability to do large scale-up at low cost, and the low maintenance requirements. Here we report a feasibility study for production of a human therapeutic protein through transplastomic transformation technology, which has the additional advantage of increased biological containment by apparent elimination of the transmission of transgenes through pollen. We show that chloroplasts can express a secretory protein, human somatotropin, in a soluble, biologically active, disulfide-bonded form. High concentrations of recombinant protein accumulation are observed (>7% total soluble protein), more than 300-fold higher than a similar gene expressed using a nuclear transgenic approach. The plastid-expressed somatotropin is nearly devoid of complex post-translational modifications, effectively increasing the amount of usable recombinant protein. We also describe approaches to obtain a somatotropin with a non-methionine N terminus, similar to the native human protein. The results indicate that chloroplasts are a highly efficient vehicle for the potential production of pharmaceutical proteins in plants.     

Purification and characterization of pituitary bovine somatotropin

Bovine somatotropin (bST) has been isolated from pituitary glands and compared in a variety of chemical analyses and bioassays with somatotropin derived from recombinant Escherichia coli. Comparison of pituitary extracts and purified bST by Western blot analysis of two-dimensional gels suggested that the immunoreactive somatotropin species present in the extract were also present in the purified material, with no significant losses or degradation as a result of the purification method. NH2-terminal sequence analysis indicated the presence of equal quantities of Ala-Phe-Pro-Ala-Met-Ser-Leu-Ser- and Phe-Pro-Ala-Met-Ser-Leu-Ser- sequences. The Met-Ser-Leu-Ser-NH2-terminal sequence, a degradation product observed in NIH standard lots, was not detected. Assay of bioactivity in a bovine liver receptor-binding assay and in a female rat growth assay showed pituitary bST and recombinant methionyl-bovine somatotropin to be equipotent. Tryptic maps and sequence analysis of pituitary-derived somatotropin suggest the presence of isoaspartate derivatization at Asp128.     

凡纳滨对虾溶菌酶基因在毕赤酵母中的分泌表达和活性检测

原核重组表达的凡纳滨对虾(Litopenaeus vannamei)溶菌酶蛋白主要以包涵体形式存在, 经变性和复性处理后活性仍较差。研究将凡纳滨对虾溶菌酶基因(Lvlyz基因)克隆至毕赤酵母分泌型表达载体pPIC9K中, 电击转化毕赤酵母GS115细胞, 经组氨酸营养缺陷培养基筛选和PCR检测获得转化子。对其进行连续甲醇诱导表达, 利用SDS-PAGE和C端携带的6×His标签,对发酵液上清进行Western blot检测, 结果表明19.3 kD左右的条带即是重组表达的溶菌酶蛋白。用溶壁微球菌平板抑菌法鉴定表达产物具有较强的抑菌能力。研究首次利用毕赤酵母真核表达系统实现对虾溶菌酶基因的可溶性表达, 并且表达产物的活性良好。       

Coastal environmental impacts on Kuwaiti fisheries

The bumper shrimp catches of 1983 and 1988, and inter-annual variability of finfish catches, prompted a study of the role played by the marine environment in fish production variability. The available short time series of physico-chemical data, beginning in 1982, showed a distinctive pattern of variability with a possible periodicity of five to six years. The two bumper shrimp years coincided with the trough of the time series of yearly averaged values for temperature, salinity and dissolved bottom oxygen. The catches for four commercially important finfish species were low in 1982 and high in 1984/1985. The trend in fish catches lagged by one year and showed an inverse relationship with shrimp production. The forces behind the formation of the special environmental conditions of 1983 and 1988 are assumed to be high discharge from the Shatt Al-Arab and less windforcing. It was not feasible to estimate the magnitude of the environmental relative to the effects of seasonal closure of the shrimp fisheries as the available time series are not sufficiently long, but such studies should eventually be undertaken.     

Utilization of shrimp industry waste in the formulation of tilapia (Oreochromis niloticus Linnaeus) feed

A rapid expansion of fisheries is demanding an adequate supply of efficient, nutritious and inexpensive fish feed, because feed contributes highly to the cost of fish production. Shrimp head, a waste product from the shrimp export industry qualifies as an economical, abundant and good quality protein source for fish feeds. In the present work, shrimp head silage powder, which contained approximately 40% protein, was used as a substitute for fish flour. Four feeds, in the form of pellets, were prepared by substituting shrimp head silage for fish flour at 0%, 33.3%, 66.6% and 100% dietary levels. Other ingredients such as corn, soy, bovine blood, cassava and corn cob flours, soy oil, vitamin premix, salt, and other components also were used in the formulation. A commercial fish feed was used as the control. The proximate composition of these feeds did not differ significantly at p>0.05, except for the protein content of the control feed, which was about 30.6% versus 35.4-36.9% protein in the other diets. No significant differences (p>0.05 level) in weight and length of juveniles fed with the different feeds during a period of 60 days were observed. In all cases, an excellent correlation (0.9950-0.9996) between weight and length of juveniles was observed. No significant difference in growth of juveniles fed on R1, R2, R3, or R4, or the control feed, was observed. Similarly, the proximate analyses of the flesh of juveniles did not present significant differences (p>0.05). The result of the study indicates that the shrimp head silage could replace fish flour as an ingredient in tilapia feed with economic advantages and without sacrificing the quality of the feed.     

Shrimp (Litopenaeus vannamei) trypsinogen production in Pichia pastoris bioreactor cultures

Recently, we engineered a Pichia pastoris Mut⁺ strain to produce and secrete recombinant Litopenaeus vannamei trypsinogen. Despite the observed toxicity of the recombinant shrimp trypsinogen to the P. pastoris cell host, when high density cell cultures in shake flasks with alanine in the induction medium were used recombinant shrimp trypsinogen could be produced. To further improve the product yield, in this work, we evaluated L. vannamei trypsinogen production in P. pastoris using a bioreactor and two recombinant P. pastoris strains with different methanol utilization (Mut) phenotypes. The effect of pH and temperature during the induction step on the trypsinogen production was also evaluated. The results indicate that temperature, pH, and Mut phenotypes influence the production of the recombinant protein, with almost no observed effect on cell growth. All cultures with the Mut⁺ strain had significant operational difficulties, such as in lowering the induction temperature, maintaining dissolved oxygen (DO) above 20%, and maintaining the methanol concentration at a constant value, and showed a decrease in metabolic activity due to trypsinogen toxicity to the cell host. In the culture with the Mut^s strain, however, the temperature, methanol concentration, and DO could be more easily controlled, the temperature could be easily decreased, and the trypsinogen caused the lowest toxicity to the host cells. After 96 h of Mut^s strain induction (pH 6 and 25°C), about 250 mg/L recombinant trypsinogen was detected in the culture medium. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2013     

Analysis of therapeutic growth hormone preparations: report of an interlaboratory collaborative study on growth hormone assay methodologies.

Recombinant DNA-derived human growth hormone (somatotropin) is widely used to treat growth hormone-deficient children. The potency of this product is determined by in-vivo bioassay in hypophysectomized rats, which is imprecise, costly and invasive, and there have been suggestions that it could safely be replaced with in-vitro or physico-chemical alternatives. In this report we present the results of a collaborative study designed to test this proposal. Somatotropin was modified by mild or severe proteolysis, mild or severe oxidation or treatment at high pH, and compared in a multi-centre collaborative study with unmodified somatotropin or with dimerized somatotropin. Participating laboratories included manufacturers and national control laboratories, and pharmacopoeial bioassays were compared with in-house in-vitro and physico-chemical bioassays. Although performing adequately with untreated somatotropin, for degraded samples the in-vivo bioassays were relatively unresponsive to changes in the growth hormone molecule. In contrast, the physico-chemical assays, in particular the reverse-phase HPLC, performed with a high degree of selectivity. We conclude that in the case of somatotropin, the in-vivo bioassay can be removed from the routine product specification with an acceptable degree of security. This however does not obviate the requirement rigorously to demonstrate biological activity in-vivo during product development, nor may the conclusions of this study be applied to other therapeutic recombinant proteins without similar collaborative investigations.     

The effect of a depot injection of recombinant bovine somatotropin on follicular development and embryo yield in superovulated Holstein heifers

Superovulated Holstein heifers (n = 32) were given a depot injection of 500 mg recombinant bovine somatotropin (rBST) or vehicle at Day 4 of the estrous cycle (7 days before the first FSH injection); at Day 11, coincidentally with the first FSH injection; or at Day 15, the time of artificial insemination. Embryos were collected nonsurgically, and the number of corpora lutea was counted by ultrasonography at Day 7 after insemination. Blood samples were taken every second day, from Day 2 of the superovulatory cycle until the day of embryo collection, and were analyzed for progesterone, somatotropin and insulin-like growth factor-1 (IGF-1). Somatotropin-treated heifers at Day 11 had a significantly higher mean number of corpora lutea than the controls (18.1 vs 13.4; P 0.63), but it was negatively correlated with progesterone (P 相似文献   

Multiple predator effects in an intertidal food web

1. We examined the effects of multiple predators from an intertidal boulder food web to test whether and how three different predator species affected the survival of a small amphipod species. 2. Predators were chosen because they differ in their foraging mode, two feeding at the bottom and in benthic refuges (nemertean and shrimp) and one in the water-column (juveniles of a fish). 3. Mortality of amphipods was not affected by nemerteans, but was high in the presence of shrimp or fish. Highest mortalities were observed in predator-combinations that contained both shrimp and fish. Amphipods responded to shrimp by escaping into the water column, while they avoided fish by remaining in the refuge. We conclude that predator-specific defence causes conflicts for prey when both shrimp and fish are present. 4. Comparing observed effects of multiple predators with expected effects revealed risk enhancement for the shrimp + fish combination. A comparison of different predictive models revealed that the multiplicative model was most appropriate, although additive models may work well under certain conditions. 5. Based on known consumption-ranges of the predators used, we conclude that nemerteans were saturated with prey while fish were far from their saturation point. A predator's functional response curve (prey consumption in relation to prey abundance) determines its impact on prey populations. This knowledge appears essential in order to predict whether prey organisms face risk enhancement, risk reduction or additive effects of multiple predators.