毕赤酵母中外源表达脂肪酰-CoA还原酶生产脂肪醇

【目的】通过在毕赤酵母Komagataella pastoris GS115中外源表达来源于霍霍巴[Simmondsia chinensis(Jojoba)]的脂肪酰-Co A还原酶Jojoba FAR,利用微生物发酵生产脂肪醇。【方法】以质粒p RL105为模板PCR扩增获得霍霍巴脂肪酰-Co A还原酶的编码基因,以p GAPZαA为载体构建重组表达质粒p GAP-far,并通过电转化法转入K.pastoris GS115,筛选转化子并发酵,气相色谱-质谱联用检测发酵产物。【结果】构建了毕赤酵母重组菌株p GAPZ-far-GS115,通过摇瓶发酵检测到脂肪醇的合成。随后在7 L规模的发酵罐上发酵验证,得到脂肪醇产量为134.74 mg/L,产率为1.22 mg/(L·h)。【结论】实现了脂肪醇在毕赤酵母中的生物合成,为工业上利用毕赤酵母生产脂肪醇奠定了一定基础。     

洛伐他汀酰基转移酶在毕赤酵母中的高效表达

目的:构建并筛选高效表达洛伐他汀酰基转移酶(Lov D)的毕赤酵母重组菌株。方法:将突变的Lov D基因克隆到毕赤酵母胞外表达质粒p PIC9K和胞内表达质粒p AO815中,将重组表达质粒电转入毕赤酵母GS115中,得到毕赤酵母重组菌株,通过摇瓶发酵筛选高酶活力的重组菌株;在此基础上,研究重组菌在5L发酵罐中的高密度发酵,并将所得酶液进行辛伐他汀催化反应。结果:p PIC9K-Lov D胞外表达重组菌的酶活是p AO815-Lov D胞内表达重组菌的3倍。筛选到酶活高的p PIC9K-Lov D-3菌株进行5L发酵罐放大实验,经过96 h的甲醇诱导表达,酶活可达609.3 U/L;发酵所得酶液冻干后进行酶功效实验,反应45 h后,其底物转化率可达96%以上。结论:构建的毕赤酵母胞外表达菌株可高效表达洛伐他汀酰基转移酶,培养液上清杂蛋白较少,有利于后续分离和纯化,为洛伐他汀酰基转移酶的工业化生产奠定了基础。     

里氏木霉内切-β-葡聚糖酶Ⅱ基因在毕赤酵母中的表达及酶学性质研究

采用外显子拼接的方法,以里氏木霉Trichoderma reesei基因组 DNA 为模板,克隆出内切-1,4-β-D-葡聚糖酶II基因egl2的全编码序列,将其插入巴斯德毕赤酵母Pichia pastoris表达载体pPIC9K中,并位于α-因子信号肽序列的下游,获得重组质粒pQY2025。重组质粒线性化后用电穿孔法导入毕赤酵母Pichia pastoris菌株GS115中,经大量筛选,获得高效分泌表达内切葡聚糖酶II的毕赤酵母工程菌株Gp2025。用甲醇诱导培养基进行摇瓶发酵,培养基中内切葡聚糖酶II的活力可达1573.0U/mL,同时对重组内切葡聚糖酶II的性质进行了初步研究。     

甘露聚糖酶基因在毕赤酵母中的表达及酶学性质研究

采用PCR的方法,以枯草芽孢杆菌Bacillus subtilis基因组 DNA 为模板,克隆出甘露聚糖酶MAN的成熟肽编码序列,将其插入巴斯德毕赤酵母Pichia pastoris表达载体pPIC9K中,并位于α-因子信号肽序列的下游,获得重组质粒pPIC9K-MAN。重组质粒线性化后用聚乙二醇法导入毕赤酵母Pichia pastoris菌株GS115中,经大量筛选,获得高效分泌表达甘露聚糖酶的毕赤酵母工程菌株MAN22。将此菌株在5 L发酵罐中进行高密度发酵,测定酶活最高达1102IU /mL,同时对重组甘露聚糖酶的性质进行了初步研究。     

特异腐质霉中性内切葡聚糖酶Ⅱ基因的克隆及表达

利用RT-PCR的方法,以特异腐质霉（Humicola insolerts）H31-3总RNA为模板,克隆到中性内切葡聚糖酶Ⅱ基因egl2的cDNA,将其插入到表达载体pGAPZαA中,重组质粒经线性化,电击转化毕赤酵母（Pichia pastoris）菌株GS115,筛选到分泌表达重组EGⅡ的毕赤酵母工程菌株。SDS-PAGE检测结果表明,重组EGⅡ在酵母中得到了特异性表达,表达产物的表观分子量约为55kD,同时对工程菌株的发酵条件和重组EGⅡ的性质进行了初步研究。     

恶性疟原虫膜蛋白Pfs25在毕赤酵母中的组成型表达

目的:Pfs25蛋白是传播阻断型恶性疟疾疫苗的侯选抗原,在毕赤酵母中表达Pfs25蛋白,并对表达产物进行鉴定。方法:参照GenBank中公布的pfs25基因序列,通过毕赤酵母喜好密码子分析人工合成目的基因;采取定向克隆策略构建重组表达质粒pfs25/pGAPZαA,经BstXⅠ线性化,电转染法转化酵母菌株GS115,在Zeocin抗性的筛选培养基上获得表达目的基因的pfs25/pGAPZαA/GS115重组酵母菌,SDS-PAGE和Western印迹检测表达产物;通过在YPD培养基上传代培养和目的基因表达,验证重组菌株的遗传稳定性。结果:在毕赤酵母中表达了Pfs25蛋白,且重组菌株遗传性质稳定。结论:为研制基于Pfs25蛋白的传播阻断型恶性疟疾疫苗奠定了基础。     

中华绒螯蟹Δ9-脂肪酸去饱和酶基因克隆和真核表达系统的构建

Δ9-脂肪酸去饱和酶是多不饱和脂肪酸合成途径的关键酶之一,可催化脂肪酸链上特定位置形成双键。本研究在已克隆到中华绒螯蟹(Eriocheir sinensis)的Δ9-脂肪酸去饱和酶(Δ9 fatty acyl-Co A desaturase,Δ9-FAD)基因基础上,为进一步了解其功能,根据中华绒螯蟹Δ9-FAD基因c DNA序列(accession number:JQ693685)以及真核表达载体p PIC3.5K,设计引物并在其上下游分别加入Bam HⅠ和Eco RⅠ酶切位点;利用反转录PCR(RT-PCR)技术,克隆其开放阅读框(open reading frame,ORF)片段,构建重组质粒p PIC3.5k-Δ9-FAD;利用电穿孔仪将经限制性内切酶SacⅠ线性化后的重组质粒p PIC3.5k-Δ9-FAD转入毕赤酵母(Pichia pastoris)GS115菌株中。经筛选与PCR验证,得到含有重组质粒p PIC3.5k-Δ9-FAD的毕赤酵母转化株。本实验成功构建了中华绒螯蟹Δ9-脂肪酸去饱和酶的毕赤酵母表达系统,为深入研究Δ9-脂肪酸去饱和酶的功能做了基础性的工作。     

特异腐质霉中性内切葡聚糖酶Ⅱ基因的克隆及表达*

利用RT-PCR的方法,以特异腐质霉(Humicolainsolens)H31-3总RNA为模板,克隆到中性内切葡聚糖酶Ⅱ基因egl2的cDNA,将其插入到表达载体pGAPZαA中,重组质粒经线性化,电击转化毕赤酵母(Pichiapastoris)菌株GS115,筛选到分泌表达重组EGⅡ的毕赤酵母工程菌株。SDS-PAGE检测结果表明,重组EGⅡ在酵母中得到了特异性表达,表达产物的表观分子量约为55kD,同时对工程菌株的发酵条件和重组EGⅡ的性质进行了初步研究。     

人白介素2受体γ链胞外区在巴斯德毕赤酵母中的表达

目的:在毕赤酵母GS115中表达重组人白细胞介素2受体γ链(rhsIL-2Rγ)胞外区。方法:用RT-PCR法从正常人淋巴细胞中获得IL-2Rγ胞外区基因;构建重组质粒pPIC9K-hsIL-2Rγ,用聚乙二醇法转入感受态GS115菌株,MD平板筛选His+转化子,用BMMY培养基诱导表达rhsIL-2Rγ;对重组蛋白进行免疫酶染色、SDS-PAGE及Western印迹鉴定。结果:克隆到目的片段,构建了重组质粒pPIC9K-hsIL-2Rγ;免疫酶染色、Western印迹等结果显示,重组质粒已成功转化GS115,并获得诱导表达的rhsIL-2Rγ。结论:在毕赤酵母GS115中表达了rhsIL-2Rγ,其蛋白条带有上移现象,分子较大,可能其糖基化过度或存在二聚体。     

利用毕赤酵母系统表达重组人生长激素的研究

为了获得重组人生长激素在毕赤酵母中高表达的菌株,按毕赤酵母基因密码子偏爱性,人工合成hGH的全基因序列.该基因被克隆到穿梭质粒pPIC9K中,PEG1000介导转入毕赤酵母GS115细胞,通过G418筛选获得高拷贝转化子.在甲醇的诱导下.实现了hGH在毕赤酵母中的成功表达.通过发酵条件的优化.发酵上清中的表达量达1537 mg/L经过超滤和两步层析,重组蛋白的得率这35%,纯度为97%,相对分子质量测定表明重组蛋白的相对分子质量与理论值相近.N-端氨基酸测序证实hGH基因在毕赤酵母中获得正确的表达.     

Recent advances in the study of Kaposi's sarcoma-associated herpesvirus replication and pathogenesis

It has now been over twenty years since a novel herpesviral genome was identified in Kaposi's sarcoma biopsies. Since then, the cumulative research effort by molecular biologists, virologists, clinicians, and epidemiologists alike has led to the extensive characterization of this tumor virus, Kaposi's sarcoma-associated herpesvirus(KSHV; also known as human herpesvirus 8(HHV-8)), and its associated diseases. Here we review the current knowledge of KSHV biology and pathogenesis, with a particular emphasis on new and exciting advances in the field of epigenetics. We also discuss the development and practicality of various cell culture and animal model systems to study KSHV replication and pathogenesis.     

The structure, reproduction and taxonomy of Vidalia and Osmundaria (Rhodophyta, Rhodomelaceae)

Comprises species occurring mostly in subtidal habitats in tropical, subtropical and warm-temperate areas of the world. An analysis of the type species, V. spiralis (Sonder) Lamouroux ex J. Agardh, a species from Australia, establishes basic characters for distinguishing species in the genus. These characters are (1) branching patterns of thalli, (2) flat blades that may be spiralled on their axis, (3) width of the blade, (4) primary or secondary derivation of sterile and fertile branchlets and (5) position of sterile and fertile branchlets on the thalli. Application of the latter two characters provides an important basic method for separation of species into three major groups. Osmundaria , a genus known only in southern Australia, was studied in relation to Vidalia , and its separation from the Vidalia assemblage is not accepted. Species of Vidalia therefore are transferred to the older genus name, Osmundaria. Two new species, Osmundaria papenfussii and Osmundaria oliveae are described from Natal. Confusion in the usage of the epithet, Vidalia fimbriala Brown ex Turner has been clarified, and Vidalia gregaria Falkenberg, described as an epiphyte on Osmundaria pro/ifera Lamouroux, is revealed to be young branches of the host, Osmundaria prolifera.     

New or rare chromosome counts in Artemisia L. (Asteraceae, Anthemideae) and related genera from Kazakhstan

Fifteen chromosome counts of six Artemisia taxa and one species of each of the genera Brachanthemum, Hippolytia, Kaschgaria, Lepidolopsis and Turaniphytum are reported from Kazakhstan. Three of them are new reports, two are not consistent with previous counts and the remainder are confirmations of very scarce (one to four) earlier records. All the populations studied have the same basic chromosome number, x = 9, with ploidy levels ranging from 2x to 6x. Some correlations between ploidy level, morphological characters and distribution are noted.     

肝癌中HBV和HCV基因和抗原的分布及意义

采用原位分子杂交方法检测HCV RNA及HBV X基因;采用免疫组织化学方法研究HCV核心抗原,非结构区C33c抗原及HBxAg在肝细胞肝癌中的定位及分布.结果表明(1)HCV RNA、HBV X基因在肝细胞肝癌组织检出率分别为40%(55/136)和82%(112/136).HCV RNA定位于癌细胞的胞浆内,阳性细胞呈散在、灶状及弥漫分布三种形式;HBV X基因在肝癌细胞中的分布呈胞浆型、核型及核浆型,阳性细胞也呈上述三种分布形式;(2)HCV C33c抗原、核心抗原在肝细胞肝癌中的阳性率为81%(133/164)及86%(141/164).C33c抗原定位于癌细胞及肝细胞的胞浆内;核心抗原既定位于癌细胞核中,又可定位于胞浆中.C33c抗原阳性细胞以灶状分布为主;而核心抗原阳性细     

Selection with two alleles and complete dominance

For a plant selection model with frequency-independent viabilities, fertilities and selfing rates, it is shown that apart from global fixation, for certain parameter combinations a protected polymorphism and facultative fixation (either allele may become fixed according to initial frequencies) may both occur. Facultative fixation requires different selling rates for the dominant and recessive type. Protection of the polymorphism requires resource allocation for male and female function. In this connection the problem of purely genetically caused population extinction is discussed.
For general frequency dependence and regular segregation, the chances for establishment of a completely recessive gene are compared to those of a completely dominant gene. It is proven that the process of establishment of the recessive gene, despite a fitness advantage, may be considerably endangered by drift effects if random mating prevails. The recessive gene may reach the same effectivity in establishment as a dominant gene, only if the recessive homozygote mates exclusively with its own type during the period of establishment.