嗜碱芽孢杆菌耐热耐碱内切木聚糖酶基因的密码子优化及在毕赤酵母中的表达

【目的】研究极端耐热耐碱的木聚糖酶,使其能够满足造纸行业中碱性高温的苛刻环境。【方法】将一种来源于嗜碱芽孢杆菌S7的耐热耐碱内切木聚糖酶基因(xyn10A)密码子进行优化,基因合成后克隆至pHBM905BDM载体上,并转化毕赤酵母GS115菌株。【结果】木聚糖酶基因(xyn10A)优化后与原始序列比对相似性为76.70%。基因合成后克隆到pHBM905BDM载体上,并成功转化毕赤酵母GS115菌株。通过交联木聚糖底物平板水解圈法初筛及摇瓶复筛得到一株产酶量较高的菌株,且其在摇瓶发酵第10天达到最高酶活力,为495U/mL。另外,糖基化分析表明该酶在毕赤酵母中表达时被糖基化修饰。【结论】密码子优化后的极端耐热耐碱木聚糖酶基因在毕赤酵母中成功表达。     

Expression and characterization of HGV E2 cDNA inPichia pastoris

A 559 base pair fragment of cDNA locating at the putative E2 region of GBV-C/HGV was inserted intoPichia pastoris expression vector pPIC9K in the reading frame of α-factor secreting signal peptide. The recombinant expression plasmid pPIC9K-E2 was introduced intoP. pastoris GS 115 with electroporation and recombined with the host genome by homological recombination. The His⁺Mut⁺ recombinant yeasts were selected and cultivated in the BMMY medium. After 3 days induction with 0.5% methanol, the target protein (E2) accumulated up to 30% of total proteins in the supernatant. The expressed E2 protein was proved possessing antigenicity and high specificity with Western blot and ELISA probed with sera from the immunized rabbits and the patients infected by GBV-C/HGV. Biography: Wang Zhuo-hua (1977-), female, Master, research direction: genetic engineering pharmaceuticals.     

Expression of human β-subunit nerve growth factor (hNGFB) in yeast Pichia pastoris and E. coli

The gene hNGFB encoding the β subunit of human nerve growth factor (hNGF) was cloned intoP. pastoris secretive expression vector pHIL-S1 andE. coli expression vector pET-15b. The recombinant hNGFB vectors pSNGF and pET15b-NGF were transformed intoP. pastoris host cell GS115 (Mut⁺, His⁻) andE. coli strain BL21 (DE3) respectively. Expression and secretion of hNGFB inP. pastoris was attempted under the direction of the AOX1 promoter and PHO1 signal sequence. The positive colonies growing on medium without histidine were further selected by PCR. The yield of rehNGFB in GS115 was about 14.4% of total cellular secretive protein. The secreted protein was immunological active on Western blotting with rabbit anti-mNGFB antibodies. The fusion protein yield of rehNGFB inE. coli BL21 (DE3) was about 10.3% of total cellular protein after IPTG induction. Western blot detection showed its immunological activity.     

牛凝乳酶基因在毕赤酵母中的表达

构建3株表达凝乳酶的重组毕赤酵母,通过甲醇诱导收集BMMY液体培养基上清后,通过Arima K方法分别对其进行酶活测试并进行酶学性质分析.结果表明,GS115/pPIC9K-chy1菌株诱导7d后,其酶活达到1.905SU/mL重组凝乳酶的最适反应温度为45℃;最适反应pH为6;在pH 4~7之间凝乳活性相对稳定;在50℃以下可以保持酶活,在55℃处理50min后,丧失60%的活性.     

发酵木糖高产乙醇树干毕赤酵母菌株的Co60诱变选育

【目的】选育能够利用木糖高产乙醇的酵母菌株。【方法】采用Co60诱变树干毕赤酵母(Pichia stipitis),筛选乙醇产量高的突变菌株,并对原始菌株、突变菌株的生长发酵特性和两个菌株对高浓度木糖、乙醇的耐受性进行比较。【结果】在YPX培养基上筛选获得1株能够高效发酵木糖的突变菌株1K-9。该菌株在50mL 15%FM发酵84h,发酵液乙醇含量最高达(51.034±0.112)g/L,比原始菌株提高10.05%;在500mL 15%FM发酵96h,乙醇含量最高达(51.390±0.119)g/L;在500mL 20%FM发酵156h,乙醇含量最高达(52.496±0.513)g/L。菌株1K-9在HSM培养基或含4%~5%乙醇的YPX培养基中生长良好,在含6%~7%乙醇的YPX培养基中生长缓慢。【结论】Co60诱变对于树干毕赤酵母(Pichia stipitis)菌株是有效的,能选育出木糖高产乙醇酵母菌株1K-9。     

产丁醇大肠杆菌工程菌的构建

【目的】为了在大肠杆菌(Escherichia coli)中导入改良的丁醇合成途径,使非生产菌株大肠杆菌具备产丁醇的能力。【方法】克隆大肠杆菌乙酰转移酶基因atoB和丙酮丁醇梭菌(Clostridium acetobutylicum)丁醇合成途径关键酶基因(crt、hbd、adhE),构建多顺反子表达质粒pSE380-atoB-adhE-crt-hbd;克隆齿垢密螺旋体(Treponema denticola)反式烯酰辅酶A还原酶基因ter,构建表达质粒pSTV29-ter,并将双质粒导入到大肠杆菌。【结果】构建的工程菌能半厌氧发酵产微量丁醇,产量为0.08g/L。【结论】大肠杆菌中的丁醇合成途径导入成功,构建了产丁醇的大肠杆菌工程菌。     

Expression and antigen activity determination of human thyroid peroxidase in silkworm and yeast

In this study, we report the expression of human thyroid peroxidase (TPO) in silkworm larvae and Pichia pastoris GS115. Recombinant TPO is sequentially purified from the hemolymph of infected silkworm larvae and yeast using a Ni-NTA resin kit. The concentration of yield of recombinant TPO is 4.87 mg per thousand larvae and 40.83 mg per liter yeast culture. However, the recombinant TPO produced in silkworm show similar binding ability with the specific anti-TPO serum to standard human TPO purified from insect cells. The lower antigen activity indicates the TPO expressed in yeast is not suitable to be used as the coating antigen in enzyme linked immunosorbent assay (ELISA). The cost of TPO expressed in B. mori is about 1/4 that of in insect cells, and the cost of TPO purified from silkworm for ELISA is only 1/8 that of TPO produced from Sf9 cells. It indicates the BmNPV-silkworm expression system is a cost-effective platform for producing TPO with high antigen activity.     

A tumor suppressor genefhit prokaryotic expression and identification

The recombinant expression vector pGEMD-fhit which contains full encoding region offhit gene was constructed. The recombinant was introduced into the BL21 (DE3) strain ofE. coli and induced by 1 mmol/L IPTG to express a 29×10³ polypeptide offhit fusion protein. And the 29×10³ protein was sensitive and specific in reaction with anti-fhit antibody in Western blot. Foundation item: Supported by the National Natural Science Foundation of China (39770373) Biography: SUN Yan (1975−), female, Master of science, Research direction: gene engineering     

Cloning and Expression of BTB/POZ-Domain from Zebrafish gcl （Germ Cell-Less）, and Appraisement of Its Polyclonal-Antibody

In this study, a recombinant pET28c-gBTB/POZ was constructed by cloning the sequence of the BTB/POZ domain of the zebrafish gcl （germ cell-less） into the expression vector pET28c, and pET28c-gBTB/POZ was transformed into BL21（DE3） pLysS strain to express the fusion protein for the preparation of antibody. Polyclonal-antibody against the GCL-BTB/POZ domain was prepared by immunizing rabbit with the fusion protein, and the Western Blot and immuno-histochemical analysis were performed to detect the quantity of the polyclonal-antibody. The result indicates that the polyclonal-antibodies were of good quantity and specification. Further studies will be performed to demonstrate the function and expression pattern of the GCL protein during the development process of zebrafish with the polyclonal-antibody.     

鸡白细胞介素-2在毕赤酵母中的表达

将鸡白细胞介素-2(ChIL-2)基因插入酵母分泌型表达载体pPIC9K 构建重组表达载体pPIC9K/ChIL-2,通过电转化构建毕赤酵母(Pichia methanolica)GS115 甲醇利用型重组表达菌株.经SDS-PAGE与Western blot分析,证实ChIL-2基因在重组GS115中成功表达出约16 ku与14 ku两条特异性条带,这与天然ChIL-2相对分子质量大小一致,提示ChIL-2可能为糖基化蛋白质.     

小球藻(Chlorella vulgaris)抗铵品系的紫外诱变选育

【目的】获得耐受高铵浓度的优良养殖小球藻(Chlorella vulgaris)品系,可以有效防止其在养殖过程中被轮虫,纤毛虫等污染生物吞食。【方法】首先对小球藻进行紫外线诱变,然后用高浓度NH_4HCO_3作为筛选压力对其进行筛选。【结果】筛选到具有生长优势的小球藻突变株B4和C6,培养12d,在NH_4HCO_3浓度为800mg/L、1 000mg/L、1 200mg/L条件下,B4和C6的生物量比出发藻株分别提高41.18%和9.54%,32.64%和29.41%,37.74%和22.85%;对比出发藻株,B4和C6的最大光能转化效率有显著提高。【结论】突变株B4和C6在高铵培养条件下更能显示生长优势,且它们的生长优势可能来自光合效率的提高。     

沼液和香蕉秆压榨汁生产单细胞蛋白菌株的筛选

由于全球蛋白质缺乏,单细胞蛋白(Single cell protein,SCP)作为一种安全的食品添加剂和饲料,越来越受到人们的重视。尽管SCP大规模生产历史已有近百年,但是对各种优良生产菌株、潜在底物以及最优发酵条件的研究仍然是世界各国研究的热点。基于此,本研究以沼液和香蕉秆压榨汁为研究对象,对能利用其生长的菌株进行筛选,为进一步的单细胞蛋白生产提供参考。实验结果表明:酵母菌、芽孢杆菌都能利用沼液和香蕉秆压榨汁生长,但香蕉秆压榨汁不易保存,因此不宜作为发酵底物;热带假丝酵母Candida tropicalis在沼液中的生长能力强于酿酒酵母Saccharomyces cerevisiae Y49和毕赤酵母Pichia pastoris GS115;芽孢杆菌在沼液中的生长无规律,且易染杂菌,不适合实际生产应用。因此,沼液作为热带假丝酵母单细胞蛋白生产的培养基,不仅能为单细胞蛋白的生产提供原料,也有利于沼液的妥善处理,实现经济效益和环境效益的双赢。     

RGD-拖丝蛋白基因的构建及其在毕赤酵母中的分泌表达

根据天然拖丝蛋白的高度重复性序列,引入与细胞黏附有关的精氨酸-甘氨酸-天冬氨酸(RGD)三肽序列,以毕赤酵母偏好的密码子化学合成RGD-拖丝蛋白基因单体,通过"头尾相连"的多聚化策略,倍加成16聚体与32聚体基因.分别将这两种多聚体与分泌型表达载体pPIC9K连接,转化毕赤酵母GS115,用G418筛选毕赤酵母重组菌.通过甲醇诱导表达,培养液上清的SDS-PAGE分析表明RGD-重组拖丝蛋白获得分泌型表达.     

苏云金芽孢杆菌aiiA基因在毕赤酵母中的分泌表达

根据GenBank中aiiA基因设计引物,以苏云金芽孢杆菌基因组为模板扩增出aiiA基因后构建出pPIC9K-aiiA重组表达载体,线性化后转化毕赤酵母GS115,获得重组工程菌GS115/pPIC9K-aiiA,以体积分数为1%的甲醇进行诱导表达.表达产物经SDS-PAGE及Western blotting分析显示表达的蛋白具有较好的免疫特异性.抗病性实验显示表达的目的蛋白具有生物学活性和良好的抗病能力.     

Cloning and Characterization of the fecC Gene Necessary for Optimal Growth under Iron-Deficiency Conditions in the Cyanobacterium Anabaena sp. PCC 7120

The fecC gene encoding a putative iron (Ⅲ) dicitrate transporte rwas cloned from nitrogen-fixing cyanobacterium Anabaena sp. PCC 7120, and inactivated. The mutant grows normally in medium with NO3^- , NH1^- or without combined nitrogen. But in iron-deficient medium, the mutant grows slowly. Photosynthetic properties were compared between the mutant and the wildtype strain, the content of photosynthetic pigments in the mutant is lower than that of the wild-type. The results of RT-PCR experiments show that the fecC gene is expressed under iron-deficient conditions, but is not expressed under iron-replete conditions. These results revealed that fecC gene product is required for optimal growth under iron-deficient conditions in Anabaena sp. PCC 7120.     

A note on m -weights of linear codes

m-weight, as a new generalization of classical Hamming weight, was discussed in this paper. A condition for the existence of linear codes of certainm-weights was given; the Singleton bound, Plotkin bound and Sphere Parking bound of Hamming weight were correspondingly generalized to them-weight. Foundation item: Supported by the National Program on Basic Sciences (973 Program, G1999075102) Biography: YUAN Yuan (1979-), female, Ph. D candidate, research directions: combination and codes.     

毕赤酵母FLD1启动子元件的克隆与测序

为了应用毕赤酵母表达某些食用蛋白或同时表达多个异源蛋白,本文以毕赤酵母GS115基因组DNA为模板,采用prime5.9程序设计了一对不等长引物,经多轮直接多聚酶链式反应,扩增获得了大小为597bp目标DNA片段;再经限制性内切酶和双脱氧末端终止法分析,其DNA片段排列顺序与EMBL发表的FLD1启动子的序列完全一致。     

解淀粉芽孢杆菌中性植酸酶基因的克隆及其在毕赤酵母中的表达

采用PCR法从解淀粉芽孢杆菌BA11中克隆到一个中性植酸酶phyc基因,将该基因克隆到毕赤酵母表达载体pPIC9K上并电转化至宿主细胞GS115后进行诱导表达。SDS-PAGE试验表明:该重组中性植酸酶在毕赤酵母宿主细胞中实现了高效分泌性表达。植酸酶活性测定结果显示阳性克隆子在诱导72h酶活性达到最高值,活性为2330U/L。     

南极假丝酵母脂肪酶B在毕赤酵母的表达及酶学性质研究

为提高南极假丝酵母脂肪酶B(Candida antarcticalipase B,CALB)的表达量,根据毕赤酵母(Pichia pastoris)密码子偏好性设计合成CALB基因,插入表达载体pPIC9K构建重组质粒pPIC9K-CALB.重组质粒转化毕赤酵母GS115,经过G418抗性筛选得到多拷贝转化子.摇瓶发酵120 h后上清液酶活力达到46 U/mL,通过金属螯合层析纯化发酵液将CALB纯化了5.23倍,比活力达到856.7 U/mg,去糖基化实验显示重组CALB比野生型CALB大5 ku.实验考察了不同反应温度、pH值和金属离子对重组CALB活性和稳定性的影响,发现重组CALB最适反应温度和pH分别为30℃和6.5,在pH 5.0~8.0之间以及40℃以下有较好稳定性,金属离子(10 mmol/L)Ca2+,Zn2+,Mn2+有助于CALB酶活力的提高,而Cu2+,Ag+,Fe3+强烈抑制酶催化反应.     

Heterologous expression of amylase gene from Saccharomycopsis fibuligera in an industrial strain of Saccharomyces cerevisiae

An α-amylase encoding gene was amplified by polymerase chain reaction fromSaccharomycopsis fibuligera and inserted into a shuttle vector YEp352, together with the yeast phosphoglycerate kinase 1 promoter and α-factor signal gene. The recombinant expression plasmid pLA8α was transformed into an industrial strain ofSaccharomyces cerevisiae Sc-11. The activity of the α-amylase produced by the transformant Sc-11-pLA8α was 6.3 U/mL and the starch utilization rate in YPS medium was 42%. The purified amylase was analyzed by SDS-PAGE, showing a molecular weight of 55×10³ protein band. Furthermore, the residual sugar, ethanol and some volatile compounds in the fermented worts under simulating brewing conditions were determined by chromatographic analyses. The fermentation characteristics of Sc-11-pLA8α were similar to that of Sc-11 and only minor changes in the concentration of flavor compounds could be observed. Foundation item: Supported by the National Tenth Five-Year Hi-Technique Project (2001BA708B05-04). Biography: LIU Zeng-ran (1964-), fenale, Ph. D., research, direction: food and biotechnology.