棉花咖啡酸-O-甲基转移酶基因的原核表达及蛋白纯化鉴定

为获得大量高纯度的GhCOMT2蛋白以便研究其功能和性质,以pMD18-GhCOMT2质粒为模板,PCR扩增GhCOMT2基因的cDNA编码区,构建原核表达载体pET-28a-GhCOMT2,经酶切鉴定并测序后转化到大肠杆菌BL21 (DE3)中进行诱导表达,并采用Western blotting方法鉴定表达产物.结果表明:在大肠杆菌BL21(DE3)菌株中成功表达了与标签蛋白融合的GhCOMT2蛋白,大小约为40.062 kD,浓度为0.62 mg/mL.重组蛋白的最佳诱导条件为:0.2 mmol/L IPTG在16℃诱导12 h.重组蛋白以可溶形式高效表达,用蛋白标签亲和层析柱(His TrapTM HP)获得纯化重组蛋白,Western blotting分析表明其能与His多克隆抗体起特异性反应.     

拟南芥中柯浩体蛋白Atcoilin的克隆、表达及纯化

旨在制备柯浩体的标志蛋白——Atcoilin蛋白,利用pET-28a与目的基因构建重组表达质粒,经DNA测序证实插入序列与设计完全一致后,将重组质粒转化大肠杆菌BL21(DE3),用IPTG进行诱导表达,产物用SDS-PAGE及Western blotting分析鉴定。通过分别改变IPTG的浓度、培养时间、培养温度等来优化Atcoilin蛋白的表达条件。表达出的重组蛋白经过镍柱、分子筛进行纯化。结果显示,原核表达载体pET28a-At1g13030成功构建,可在大肠杆菌BL21(DE3)中诱导表达,得到相应的重组蛋白经Western blotting鉴定正确。在IPTG浓度为0.7 mmol/L,18℃培养20 h的条件下,目的蛋白表达量最高。经过SDS-PAGE分析鉴定,过镍柱、分子筛后得到的重组蛋白纯度较高。     

水牛MyD88cDNA的克隆与原核表达

采用RT-PCR方法从水牛外周血白细胞总RNA中扩增出髓样分化因子88 (mydoid differentiation factor 88,MyD88) cDNA序列,PCR产物分离纯化后,与pMD20-T载体连接,重组质粒经PCR、酶切鉴定后测序,并进行生物信息学分析;构建pET28a-MyD88表达载体,并将其转化至E.coli BL21 (DE3),经IPTG诱导表达后,进行SDS-PAGE、镍柱亲和层析纯化和Western blotting分析.结果显示,克隆到的水牛MyD88 cDNA全长为1 189 bp,含有1个891 bp的开放阅读框,编码296个氨基酸,理论等电点为5.65.经IPTG诱导表达后,得到一个带His·Tag的约39 kD的重组融合蛋白.用抗His单克隆抗体进行Western blotting,得到1条约39 kD特异性抗体结合带,表明水牛MyD88原核表达载体成功构建并表达.本研究为进一步开展水牛MyD88的结构功能分析奠定了基础.     

重组人可溶性BAFF蛋白的原核表达、纯化及鉴定

目的:在大肠杆菌中表达人B细胞活化因子可溶性胞外域134~285(rhsBAFF134-285)蛋白,并进行纯化与鉴定。方法:重组表达质粒pET-32ammrhsBAFF在大肠杆菌Rosetta-gami B(DE3)中于16℃下进行IPTG诱导表达。经His亲和层析纯化得到融合蛋白后进行TEV蛋白酶酶切切除Trx融合标签,进而纯化得到rhsBAFF目的蛋白,SDS-PAGE、Western Blot和ELISA鉴定纯化产物。结果:Trx-rhsBAFF融合蛋白相对分子量约31000,16℃表达时主要以包涵体形式表达,上清中也有部分表达。融合蛋白纯化后经酶切再纯化得到相对分子量约17000、纯度95%以上的rhsBAFF目的蛋白,鉴定可被小鼠抗rhBAFF单克隆抗体和小鼠抗rhBAFF多抗血清特异性识别。结论:成功原核表达并纯化得到rhsBAFF蛋白,为进一步开发用于研究人类自身免疫性疾病的BAFF检测试剂盒奠定基础。     

FMDV整联蛋白受体b1亚基的克隆及其配体黏附区多抗的制备

采用RT-PCR技术从牛气管组织扩增出2400 bp的b1基因, 回收纯化连入PGEM-T载体, 测序。用Expasy软件对b1基因的抗原性进行分析, 选取胞外区334~861 bp的配体结合区与6×His融合, 在大肠杆菌中大规模诱导表达, 并经Ni2+亲和柱层析纯化。通过SDS-PAGE鉴定后, 应用纯化蛋白免疫新西兰家兔, 获得效价在1:12 800以上的多抗, Western blotting鉴定表明此抗体可特异性的与表达的融合蛋白作用。     

FMDV整联蛋白受体b1亚基的克隆及其配体黏附区多抗的制备

采用RT-PCR技术从牛气管组织扩增出2400 bp的b1基因, 回收纯化连入PGEM-T载体, 测序。用Expasy软件对b1基因的抗原性进行分析, 选取胞外区334~861 bp的配体结合区与6×His融合, 在大肠杆菌中大规模诱导表达, 并经Ni2+亲和柱层析纯化。通过SDS-PAGE鉴定后, 应用纯化蛋白免疫新西兰家兔, 获得效价在1:12 800以上的多抗, Western blotting鉴定表明此抗体可特异性的与表达的融合蛋白作用。     

酿酒酵母RAVE复合物的155kD亚基Rav1p的克隆及其在大肠杆菌中的表达纯化

以酿酒酵母基因组DNA为模板,根据CenBank上公布的酿酒酵母Ravlp基因(rav1)序列和表达裁体特性设计 特异性引物,PCR扩增得到4 074 bp的DNA片段,将PCR产物和原核表达栽体pET28a(+)同时进行双酶切;双酶切后的PCR产物和表达栽体进行连接,构建成重组质粒pET28a-ravl.再将pET28a-ravl转化到BL21( DE3)感受态细胞中.经IPTG 16℃低温诱导40h表达His-tag融合的Ravlp.诱导后的菌体进行超声波破碎,然后用GE healthcare公司的AKTA蛋白纯化仪和His Trap HP I mL亲和层析柱纯化目的蛋白.SDS-PAGE电泳分析和Western blot分析显示在155 kD有明显的条带,成功实现了Ravlp在大肠杆菌中的表达纯化.     

FMDV整联蛋白受体β1亚基的克隆及其配体黏附区多抗的制备

采用RT-PCR技术从牛气管组织扩增出2400 bp的β1基因, 回收纯化连入PGEM-T载体, 测序.用Expasy软件对β1基因的抗原性进行分析, 选取胞外区334～861 bp的配体结合区与6×His融合, 在大肠杆菌中大规模诱导表达, 并经Ni2+亲和柱层析纯化.通过SDS-PAGE鉴定后, 应用纯化蛋白免疫新西兰家兔, 获得效价在1:12 800以上的多抗, Western blotting鉴定表明此抗体可特异性的与表达的融合蛋白作用.     

猪盖他病毒衣壳蛋白的原核表达、纯化和多克隆抗体的制备

原核表达猪盖他病毒(Getah virus)衣壳蛋白(Cap)并制备多克隆抗体。设计一对特异性引物,从含有Cap基因的pT-Cap质粒中扩增全长Cap基因,克隆至携带有His标签的原核表达载体pColdⅠ中,通过PCR、酶切鉴定和序列测定后,重组质粒pCold-Cap转化大肠杆菌Rosetta 2,IPTG诱导后SDS-PAGE和Western blot鉴定融合蛋白;蛋白经镍柱纯化后切胶免疫Balb/c小鼠,制备多克隆抗体。实验表明:经终浓度0.1mmol/L IPTG 15℃诱导24h后,Cap基因在Rosetta 2中获得高效表达,表达量占菌体蛋白的40.2%,SDS-PAGE显示融合蛋白相对分子质量为32.3kD。Western blot显示制备的鼠源抗血清可以与融合蛋白发生反应,有明显的特异性条带。猪盖他病毒衣壳蛋白原核表达成功,制备的多抗可以识别Cap蛋白。     

非洲猪瘟病毒CP204L基因的原核表达与单抗制备

由非洲猪瘟病毒(ASFV)引起的非洲猪瘟(ASF)给我国养猪业带来了不可估量的经济损失,严重阻碍了我国养猪业的发展,研发ASFV快速诊断试剂是目前最重要的内容之一。CP204L基因编码ASFV结构蛋白p30。本研究以克隆ASFV的CP204L基因为基础,通过基因重组技术,加入His标签,将构建的重组质粒命名为pET-28a-CP204L。将重组质粒转化至大肠杆菌BL21(DE3)感受态细胞,37℃经1mmol/L异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达6h,表达蛋白进行SDS-PAGE鉴定和Western Blot检测。重组蛋白纯化后免疫小鼠制备筛选单克隆抗体,Western Blot和IFA验证单抗的结合特异性。结果表明,重组的pET-28a-CP204L诱导后表达蛋白为30kD,以不可溶性包涵体形式存在;表达蛋白利用His标签进行纯化,获得纯化蛋白2mg,单克隆抗体筛选获得5株IgG亚型的ASFV p30蛋白的单抗,且均具有良好的结合活性。本研究为发展ASFV检测方法提供了基础。     

Coccystes undoxylophus

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Flavonoids of Astilbe and Rodgersia compared to Aruncus

The flavonoid profiles of Astilbe (four taxa studied) and Rodgersia (two taxa studied) are based on simple flavonol glycosides. Astilbe has 3-O-mono-, 3-O-di-, and 3-O-triglycosides of kaempferol, quercetin, and myricetin, while Rodgersia has only mono- and diglycosides of kaempferol and quercetin. Astilbe×arendsii was also shown to accumulate dihydrochalcone glycosides. The flavonoid profile of Rodgersia is the simplest recorded so far in the herbaceous Saxifragaceae. The flavonoids of two species of Aruncus were shown to be based upon kaempferol and quercetin 3-O-mono- and 3-O-diglycosides. One of the species also exhibited an eriodictyol glycoside. The triglycoside differences were not considered important, but the differences in myricetin occurrences were taken as evidence against derivation of Saxifragaceae from an Aruncus-like ancestor. Should such an event be proposed, however, serious consideration would have to be given to the current pattern of myricetin occurrence in the two families.     

Biogeography of Hysterangiales (Phallomycetidae, Basidiomycota)

To understand the biogeography of truffle-like fungi, DNA sequences were analysed from representative taxa of Hysterangiales. Multigene phylogenies and the results of ancestral area reconstructions are consistent with the hypothesis of an Australian, or eastern Gondwanan, origin of Hysterangiales with subsequent range expansions to the Northern Hemisphere. However, neither Northern Hemisphere nor Southern Hemisphere taxa formed a monophyletic group, which is in conflict with a strictly vicariant scenario. Therefore, the occurrence and importance of long-distance dispersal could not be rejected. Although a pre-Gondwanan origin of Hysterangiales remains as a possibility, this hypothesis requires that Hysterangiales exist prior to the origin of the currently recognized ectomycorrhizal plants, as well as the arrival of mycophagous animals in Australia. This also requires that a basal paraphyletic assemblage represents parallel evolution of the ectomycorrhizal symbiosis, or that Hysterangiales was mycorrhizal with members of the extinct flora of Gondwana. Regardless, models for both ancient and more recent origins of Hysterangiales are consistent with truffle-like fungi being capable of transoceanic dispersal.     

Ester production in E. coli and C. acetobutylicum

Genetic engineering has improved the product yield of a variety of compounds by overexpressing, inactivating, or introducing new genes in microbial systems. The production of flavor-enhancing ester compounds is an emerging area of heterologous gene expression for desired product yield in Escherichia coli. Isoamyl acetate, butyl acetate, ethyl acetate, and butyl butyrate are reported here to be produced by expressing Saccharomyces cerevisiae genes ATF1 or ATF2 and the strawberry gene SAAT in E. coli when the appropriate substrates are provided. Increasing the concentration of alcohol added to the reaction generally resulted in increased ester production. ATF1 expression was found to produce more isoamyl acetate and butyl acetate than ATF2 expression or SAAT expression in the strains and culture conditions examined. Additionally, SAAT expression resulted in greater isoamyl acetate and butyl acetate production than ATF2 expression. Butyl butyrate is produced by cell-free extracts of E. coli harboring SAAT but not ATF1 or ATF2.     

The role of the genesnrf EFG andccmFH in cytochromec biosynthesis inEscherichia coli

It has been suggested that two groups ofEscherichia coli genes, theccm genes located in the 47-min region and thenrfEFG genes in the 92-min region of the chromosome, are involved in cytochromec biosynthesis during anaerobic growth. The involvement of the products of these genes in cytochromec synthesis, assembly and secretion has now been investigated. Despite their similarity to other bacterial cytochromec assembly proteins, NrfE, F and G were found not to be required for the biosynthesis of any of thec-type cytochromes inE. coli. Furthermore, these proteins were not required for the secretion of the periplasmic cytochromes, cytochromec ₅₅₀ and cytochromec ₅₅₂, or for the correct targeting of the NapC and NrfB cytochromes to the cytoplasmic membrane. NrfE and NrfG are required for formate-dependent nitrite reduction (the Nrf pathway), which involves at least twoc-type cytochromes, cytochromec ₅₅₂ and NrfB, but NrfF is not essential for this pathway. Genes similar tonrfE, nrfF andnrfG are present in theE. coli nap-ccm locus at minute 47. CcmF is similar to NrfE, the N-terminal region of CcmH is similar to NrfF and the C-terminal portion of CcmH is similar to NrfG. In contrast to NrfF, the N-terminal, NrfF-like portion of CcmH is essential for the synthesis of allc-type cytochromes. Conversely, the NrfG-like C-terminal region of CcmH is not essential for cytochromec biosynthesis. The data are consistent with proposals from this and other laboratories that CcmF and CcmH form part of a haem lyase complex required to attach haemc to C-X-X-C-H haem-binding domains. In contrast, NrfE and NrfG are proposed to fulfill a more specialised role in the assembly of the formate-dependent nitrite reductase.     

Multiple invasions of Gypsy and Micropia retroelements in genus Zaprionus and melanogaster subgroup of the genus Drosophila

Background  

The Zaprionus genus shares evolutionary features with the melanogaster subgroup, such as space and time of origin. Although little information about the transposable element content in the Zaprionus genus had been accumulated, some of their elements appear to be more closely related with those of the melanogaster subgroup, indicating that these two groups of species were involved in horizontal transfer events during their evolution. Among these elements, the Gypsy and the Micropia retroelements were chosen for screening in seven species of the two Zaprionus subgenera, Anaprionus and Zaprionus.     

Chemical composition, in situ ruminal degradability and post-ruminal disappearance of dry matter and crude protein from the halophytic plants Kochia scoparia, Atriplex dimorphostegia, Suaeda arcuata and Gamanthus gamacarpus

Samples of Kochia (K. scoparia), Atriplex (A. dimorphostegia), Suaeda (S. arcuata) and Gamanthus (G. gamacarpus) were collected and analyzed for chemical composition including crude protein (CP), ether extract (EE), ash, neutral detergent fiber (NDFom), acid detergent fiber (ADFom), non-protein N (NPN), Ca, P, Na, K, Cl, Mg, Fe, Cu and Se. In addition, in situ ruminal degradability and post-ruminal disappearance of dry matter (DM) and CP of the samples using a mobile bag technique were determined. Results indicate that the chemical composition of Kochia and Atriplex was notably different from those of Suaeda and Gamanthus. All of these halophytic plants had high concentrations of Na, K, Cl, Cu and Se, and low levels of Ca, P and Mg. The rapidly degradable fractions of DM and CP (g/g) of Kochia (0.31 and 0.35, respectively) and Atriplex (0.39 and 0.50, respectively) were lower than for Suaeda (0.53 and 0.55, respectively) and Gamanthus (0.56 and 0.66, respectively). Ruminal DM and CP disappearance of Kochia (444 and 517 g/kg, respectively) and Atriplex (472 and 529 g/kg, respectively) were lower (P<0.05) than those of Suaeda (553 and 577 g/kg, respectively) and Gamanthus (663 and 677 g/kg, respectively) (P<0.05) using the mobile bag technique. Suaeda had the lowest (P<0.05) NDFom and ADFom disappearance (214 and 232 g/kg, respectively) in the rumen. Kochia scoparia and Atriplex dimorphostegia have more beneficial chemical nutritive components and digestible values versus Suaeda arcuata and Gamanthus gamacarpus.     

Aquila pennata undminuta

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