SPA-Luc嵌合基因的构建及其融合蛋白活性检测

目的:葡萄球菌A蛋白-荧光素酶(SPA-Luc)原核表达载体的构建,表达,纯化,并对其生物学效应进行初步研究。方法:PCR扩增SPA和Luc基因,并连接到pET28a(+)中,构建原核表达质粒。构建正确的重组质粒转化至E.coliBL21(DE3),IPTG诱导蛋白表达,SDS-PAGE及Western Blot鉴定,同时用Ni-NTA亲和层析法纯化SPA-Luc蛋白,最后用荧光素酶试剂盒和ELISA检测融合蛋白的生物学活性。结果:成功构建重组质粒pET28a(+)-SPA、pET28a(+)-Luc和pET28a(+)-SPA-Luc,SDS-PAGE及Western Blot证明正确表达了重组蛋白,并获得了纯化的融合蛋白SPA-Luc。荧光素酶试剂盒检测发现该蛋白仍能与IgG结合,并且与兔和鼠的IgG有较高亲和性。ELISA显示SPA-Luc蛋白替代常规二抗检测抗原,具有更高的敏感性。结论:成功的克隆、表达、纯化的SPA-Luc蛋白可用于抗原抗体的检测,且比常规二抗具有更高的敏感性,为进一步研究其在免疫学中的应用奠定了实验基础。     

人纤溶酶原饼环区5(hPK5)基因的分泌型表达

构建人纤溶酶原饼环区5(hPK5)基因的原核可溶性表达载体并进行表达和纯化,获取大量高纯度、具有生物活性的hPK5蛋白。以纤溶酶原cDNA为模板,PCR扩增了hPK5基因,经过适当酶切后构建表达载体pET22b(+)hPK5,转入大肠杆菌BL21(DE3)进行表达并经组氨酸亲和层析获得纯化。带有重组质粒pET22b(+)hPK5的大肠杆菌经IPTG诱导后以可溶性形式表达16kDa的蛋白,其表达量占菌体总蛋白的30%以上,纯化后目的蛋白纯度达95%以上,Western印迹表明重组蛋白具有Histag抗原活性。构建了pET22b(+)hPK5重组质粒并成功地在大肠杆菌中获得可溶性表达,为获得大量hPK5基因工程产品奠定了实验基础。     

牛γ-干扰素在大肠杆菌中的表达及抗原性鉴定

目的在大肠杆菌中高效表达牛γ-干扰素（bovine interferon-γ,BovIFN-γ）,并对其生物活性进行初步鉴定。方法依据GenBank上基因序列人工合成BovIFN-γ基因,PCR方法扩增该基因,将其插入PET-28a载体构建原核表达质粒,转化大肠杆菌BL21中,在IPTG诱导下表达BovIFN-γ,并进行Western blot鉴定。Ni-NTA亲和层析法和电洗脱方法纯化表达的重组蛋白,用Western blot和商品化的BovIFN-γ检测试剂盒进行重组蛋白的抗原性检测。结果成功构建了BovIFN-γ原核表达载体PET-28a-BIFN-γ,并在大肠杆菌中高效表达,表达蛋白约占菌体总蛋白的32%,表达产物主要以可溶性形式存在于菌体裂解液上清中;重组蛋白可与BovIFN-γ单克隆抗体反应,Ni-NTA亲和层析法纯化的重组蛋白抗原活性比电洗脱方法纯化的抗原活性高。结论在大肠杆菌中成功表达了可溶性的BovIFN-γ蛋白,可与BovIFN-γ单抗发生反应,纯化的重组蛋白具有良好的反应原性。     

小鼠β-防御素30的原核表达和纯化

目的:在原核细胞中表达小鼠β-防御素30(DEFB30),并对表达产物进行鉴定和纯化。方法:用RT-PCR方法扩增小鼠Defb30的cDNA序列,将2个拷贝的cDNA序列串联连入原核表达载体pET28(a),构建重组表达载体pET28(a)-Defb30,并将重组表达载体转化至大肠杆菌Rosetta(DE3),IPTG诱导表达,以Western印迹分析表达产物His-DEFB30,用Ni-NTA亲和柱纯化融合蛋白。结果:构建了Defb30基因的原核表达载体,经IPTG诱导,相对分子质量约15×103的融合蛋白获得表达,Western印迹分析证实此蛋白即为目的蛋白,经Ni-NTA柱亲和纯化,获得了高纯度的融合蛋白His-DEFB30。结论:获得了在大肠杆菌中表达的DEFB30,为研究该蛋白的免疫避孕效果、抗菌活性奠定了基础。     

人FGF21原核表达载体的构建及重组蛋白表达

构建人FGF21(fibroblast growth factor,FGF)cDNA的原核表达载体并诱导其重组蛋白表达。提取人肝脏总RNA后,经RT-PCR扩增获得目的片段,构建其T载体进行保存。再构建重组原核表达载体pET-28a(+)-h FGF21,重组质粒转化至大肠杆菌菌株BL21(DE3)中,在IPTG诱导下得到可溶性表达,采用亲和层析法纯化表达产物后,进行Western blot鉴定。成功构建重组质粒pET-28(+)-hFGF21,对其进行可溶性表达后成功纯化出his-hFGF21,经Western blot鉴定该融合蛋白可与FGF21抗体特异性结合。成功构建pET-28(+)-hFGF21,并可溶性表达his-hFGF21蛋白。     

重组骆驼源单域抗体的原核表达及纯化

目的:原核表达重组骆驼源单域抗体并纯化。方法:将雌二醇特异性的单域抗体基因片段克隆到pET32a载体中,经IPTG诱导表达后对形成的包涵体进行纯化,利用间接ELISA法检测获得的单域抗体的活性,并以孕酮、雌酮和雌三醇为对照,鉴定雌二醇单域抗体的特异性。结果:重组雌二醇单域抗体的相对分子质量约为34×103,通过大肠杆菌BL21(DE3)-p ET32a构建的原核表达体系实现了雌二醇单域抗体的高水平表达,并通过包涵体纯化获得了可溶性的高纯度单域抗体抗体,经间接ELISA检测,该雌二醇特异性抗体的50%抑制浓度(IC50)为20.03 ng/mL,对孕酮和雌酮2种化合物的交叉反应率分别为29.78%和1.63%,特异性较好。结论:通过构建原核表达体系,可以获得功能性的骆驼源单域抗体。     

巴尔通体（Bartonella tribocorum）P26蛋白原核重组表达研究

目的 构建巴尔通体表面蛋白p26基因的原核重组表达载体,表达和纯化重组表达蛋白P26,并鉴定其抗原性.方法 利用PCR方法从巴尔通体B.tribocorum厦门分离株的基因组DNA中扩增出p26蛋白基因,并将该基因的编码区克隆到pGEX-4T-1表达载体中,从而构建GST-p26融合蛋白原核重组表达载体.将表达载体转化大肠埃希菌（E.coli DH5α）,诱导表达GST-p26,并运用亲和层析技术纯化蛋白.通过蛋白质斑点印迹试验和间接ELISA法检验GST-p26是否具有抗原性.结果 成功构建了p26的原核表达载体,该表达载体可在E.coli DH5α中大量表达GST-p26,原核表达的GST-p26能够与感染动物血液样本发生特异性免疫反应.结论 原核重组表达的GST-p26可作为潜在的抗原,应用于巴尔通体的血清学检测.     

蜱传脑炎病毒TBE-E-D3抗原的原核表达纯化及ELISA方法鉴定

目的:构建蜱传脑炎病毒TBE-E-D3抗原的融合蛋白原核表达质粒GSTpET-30a(+)/TBE-E-D3,在大肠杆菌中诱导表达并用亲和层析法纯化,以获得特异性诊断抗原。方法:根据目的基因序列设计PCR引物,利用基因克隆技术构建重组质粒,转入大肠杆菌DH5α后经酶切鉴定,将测序正确的重组质粒转入大肠杆菌BL21(DE3),IPTG诱导表达后进行亲和层析法纯化,SDS-PAGE分析其表达情况和纯度,间接ELISA法鉴定重组蛋白的特异性和灵敏度。结果:TBE-E-D3基因目的片段以正确的读框插入载体GSTpET-30a(+),通过IPTG诱导在大肠杆菌中正确表达,亲和纯化得到较高纯度的蛋白质;间接ELISA法证明抗原特异性和灵敏度良好,送检的15份阳性患者血清样本中检出10份阳性结果,40份健康人血清样本中检出1份假阳性结果。结论:获得的His-TBE-E-D3融合蛋白具有良好的抗原性,可作为森林脑炎病毒特异性血清学诊断的备选抗原之一。     

毛白杨PtoeIF5A4的原核表达、纯化及鉴定

为获取大量高纯度的毛白杨PtoeIF5A4蛋白以便研究其特性和生物学功能,以pGEM-T Easy-PtoeIF5A4质粒为模板,PCR扩增毛白杨PtoeIF5A4基因的cDNA编码区,测序正确后,经酶切将其插入到原核表达载体pET28a上,然后将表达载体转入大肠杆菌表达菌株BL21(DE3),经不同浓度IPTG诱导培养后发现,0.1 mmol/L IPTG,28℃诱导4 h时目的蛋白最多,且该重组蛋白主要以可溶性的形式存在。采用亲和纯化从可溶性蛋白中纯化到大小为18 kD的蛋白条带。Western blotting分析发现亲和纯化所得蛋白可以与His单克隆抗体发生免疫交叉反应,表明纯化所得蛋白是PtoeIF5A4重组蛋白。     

人Nek2蛋白原核表达纯化及其多克隆抗体制备

目的:通过原核表达系统表达人Nek2蛋白,优化表达条件并纯化Nek2蛋白,制备抗Nek2多克隆抗体。方法:将Nek2基因片段构建到原核表达载体pET30a(+)上,转化大肠杆菌BL21(DE3);加入诱导剂IPTG诱导表达,对诱导温度、诱导剂IPTG终浓度、诱导时间等条件进行优化;利用12%SDS-PAGE后250mmol/L KCl染色切胶纯化蛋白质,将纯化后的Nek2蛋白进行质谱鉴定;纯化Nek2蛋白免疫BALB/c小鼠制备多克隆抗体,运用ELISA、Western blot和免疫荧光实验检测多克隆抗体效价和特异性。结果:构建了pET30a(+)-Nek2重组原核表达质粒,诱导的重组人Nek2蛋白主要以包涵体的形式存在;蛋白质的最适诱导表达条件为28℃,180r/min条件下加入终浓度为0. 2mmol/L IPTG诱导32h;质谱分析纯化后的蛋白质为Nek2蛋白,最终获得浓度为1. 35mg/ml纯化后的Nek2蛋白;纯化蛋白免疫小鼠,多克隆抗体效价大于1∶243 000,且具有良好的抗原特异性;免疫荧光实验显示Nek2主要定位于细胞质和细胞核。结论:利用重组人Nek2蛋白获得具有良好抗原特异性的抗Nek2多克隆抗体。     

Molecular cloning and expression of a novel CYP26 gene (cyp26d1) during zebrafish early development

Proper restriction of retinoid signaling by Cyp26s is essential for development of vertebrate embryos while inappropriate retinoid signaling can cause teratogenesis. Here, we report cloning and expression analysis of a novel cyp26 gene (cyp26d1) isolated from zebrafish. The predicted protein encoded by cyp26d1 consists of 554 amino acids. It exhibits 54% amino acid identity with human Cyp26C1, 50% with zebrafish Cyp26B1 and 38% with zebrafish Cyp26A1. Whole-mount in situ hybridization shows that cyp26d1 is first expressed in sphere stage, then disappears at 50% epiboly and resumes its expression at 75% epiboly. During segmentation period, cyp26d1 message is found at presumptive hindbrain. Double in situ hybridization with krox20 and cyp26d1 reveals that cyp26d1 is expressed in presumptive rhombomere 2-4 (r2-r4) at 2-somite stage. At 3-somite stage, cyp26d1 gene is expressed in r6 and pharyngeal arch (pa) one in addition to its expression at r2 and r4. At 6-somite stage, cyp26d1 message is present in continuous bands at r2-r6 and in pa1. This expression pattern is maintained from 10-somite stage through 21-somite stage except that the expression level is greatly reduced at r2 and r4. At 21-somite stage, cyp26d1 is also found in a group of cells in telencephalon and diencephalons. At 25-31h post-fertilization (hpf), the zebrafish cyp26d1 expression domain is extended to eyes, otic vesicles and midbrain in addition to its expression in hindbrain, telencephalon, diencephalons, and pharyngeal arches. At 35-48hpf, the expression of cyp26d1 is mainly restricted to otic vesicles, pharyngeal arches and pectoral fins and the expression level is greatly reduced.     

Ultrastructure of carotenoid mutant strain R-26 of Rhodopseudomonas sphaeroides

Stained thin-sections and freeze-fractured preparations of the carotenoid-less mutant strain R-26 of Rhodopseudomonas sphaeroides grown photosynthetically revealed 2 morphological kinds of intracellular membrane systems- spherical vesicles distributed throughout the cytoplasm and lamellae confined to the periphery of the cell. The lamellar membranes appeared to be large, flattened vesicles.Non-Standard Abbreviations R Rhodopseudomonas - E exoplasmic-half - P protoplasmic-half     

Plantlets from encapsulated micropropagated buds of M.26 apple rootstock

In order to be considered usable as synthetic seeds, encapsulated explants sown underin vitro orex vitro conditions must be able to produce whole plantlets. Ninety percent of non-encapsulated M.26 apple rootstock single nodes produced a plantlet (i.e., a well-formed shoot with a root system) after 30 days of culturein vitro if the explants were previously given a 24-hour treatment with 24.6 μM IBA and 15 g 1⁻¹ sucrose in darkness. In contrast, when the single nodes were encapsulated in a calcium-sodium alginate bead immediately after the same treatment only 10% of the encapsulated explants formed a plantlet. Addition of growth regulators to the artificial endosperm and culture of the single nodes for root primordia initiation for 3, 6 or 9 days in darkness before encapsulation allowed production of 58%, 60% and 66% of plantlets, respectively.     

玉米赤霉烯酮脱毒菌PA26-7的分离鉴定及其应用效果评价

【背景】玉米赤霉烯酮(zearalenone,ZEN)是广泛污染粮谷类作物的一种雌激素类真菌毒素,不仅给农业经济带来巨大损失,还能通过食物链对人和动物健康造成危害。【目的】从微生态制剂中筛选获得能够高效降解玉米赤霉烯酮的菌株,优化其脱毒条件,测定其在饲料中的实际脱毒效果及对饲料中植酸、维生素含量变化的影响。【方法】从微生态制剂中分离出玉米赤霉烯酮降解菌,通过细胞计数试剂盒-8 (cell counting kit-8, CCK-8)测定菌株降解玉米赤霉烯酮产物的细胞毒性和雌激素活性,通过高效液相色谱法测定分离株在培养基和饲料中的解毒效果,以及分离株在霉变的豆粕、麸皮和成品饲料中固态发酵前后维生素的含量变化,通过三氯化铁比色法测定饲料脱毒前后植酸的含量变化。【结果】从微生态制剂中筛选出一株通过分泌胞外酶高效降解玉米赤霉烯酮的贝莱斯芽孢杆菌(Bacillus velezensis) PA26-7,该菌株在培养基起始pH 4.0-8.0、培养温度25-60℃条件下均可降解玉米赤霉烯酮,产物的细胞毒性和雌激素活性均较ZEN弱。PA26-7经固态发酵72h后,饲料原料(豆粕和麸皮)及霉变的成品鸡...     

Walking pattern of Trichogramma nubilale Ertle & Davis (Hymenoptera; Trichogrammatidae) on various surfaces

The leaf surfaces on which mass-reared Trichogramma spp. are released for augmentative biological control may be an important factor effecting the success of these releases. We observed and recorded the walking pattern of Trichogramma nubilale females on leaves of Zea mays, Canna × generalis (Canna lilly), Silphium perfoliatum (cup plant), Abutilon theophrasti (velvetleaf), Schizachyruim scoparium (little bluestem), a smooth and a fuzzy polyester material, and waxed paper. For each surface type, a total of 10 mated, 1–2 days old, naive and fed females were observed and their walking path was traced. Females walked fastest on waxed paper and leaves of S. scoparium and slowest on leaves of A. theophrasti and the fuzzy material. Turning rates were high on Canna × generalis and waxed paper. In general, walking speed was negatively affected by the presence and density of trichomes and possibly the lack of leaf veins. The effect of surface structures, such as trichomes is likely to scale to the body size of the searching parasitoid. Parasitoids can walk over short trichomes, but short directionally pointed trichomes (as on S. scoparium) can guide the search paths in certain directions. The effects of long trichomes may depend on trichome density relative to the parasitoid body length. When trichome density is on the same order of magnitude as 1/L² (Z. mays), wasps will stand between trichomes, and will frequently run into trichomes, which will retard walking speeds and increase turning.     

Genetic and fine structure analysis of unc-26(IV) and adjacent regions in Caenorhabditis elegans

Summary The genetic organization of unc-26(IV) and adjacent regions was studied in Caenorhabditis elegans. We constructed a fine structure genetic map of unc-26(IV), a gene that affects locomotion and pharyngeal muscle movement but not muscle structure. Eleven alleles were positioned relative to each other recombinationally and were classified according to phenotypic severity. The unc-26 gene spans at least 0.026 map units, which is exceptionally large for a C. elegans gene. All but one allele, e205, are amorphic alleles. Interestingly, e205 is hypomorphic but also suppressible by the amber suppressor sup-7. Nineteen lethal mutations in the unc-26 region were isolated and characterized. The unc-26 region is subdivided into four zones by five deficiency breakpoints. These mutations fall into 15 complementation groups. The stages of development affected by these mutations were determined.     

Molecular characterization and gene expression of lhcb5 gene encoding CP26 in the light-harvesting complex II of Chlamydomonas reinhardtii

We isolated and sequenced a cDNA clone encoding a minor chlorophyll a/b-binding protein, CP26, which is associated with the light-harvesting complex II of Chlamydomonas reinhardtii. Protein sequences of internal peptide fragments from purified CP26 were determined and used to identify a cDNA clone. The 1.1 kb lhcb5 gene codes for a polypeptide of 289 amino acids with a predicted molecular weight of 30713. The lhcb5 gene product could reconstitute with chlorophylls and xanthophylls to form a green band on a gel. Although the expression of many lhcb genes are strictly regulated by light, the lhcb5 gene was only loosely regulated. We propose that a plant acclimatizes itself to the light environment by quantitatively and qualitatively modulating the light-harvesting complex. Characterization of the primary structure and the implications of its unique expression are discussed.     

Structural constraints in expansion segments from a midge 26S rDNA

DNA sequences representing approximately 40% of the large-subunit rRNA gene from the lower dipteran Chironomus thummi were analyzed. Once aligned with their Drosophila counterparts, sequence and base content comparisons were carried out. Sequence identity was found to be high overall, except for six regions that displayed a local bias in nucleotide composition toward AT. These regions were identified as expansion segments D3, D4, D5, D6, D7a, and D12. Besides base sequence divergence, differences in length were observed between the respective variable domains of the two species, particularly for D7a. Prediction of secondary structure showed that the folding of the Chironomus expansion segments analyzed is in agreement with the general patterns proposed for eukaryotic LSU rRNA. The comparison with Drosophila revealed also that the Chironomus secondary structures of the variable domains are supported by multiple compensatory substitutions or even compensatory insertions. Chironomus D7a displayed an unusual structural feature with respect to the insect D7a models that have been inferred up to now. The structural constraint observed in the expansion segments of Diptera so distantly related as midges and Drosophila suggests that these regions contribute to some functional role. Concerning the D7a of insects so far analyzed, there can be, in addition to a conserved secondary structure, a nucleotide composition constraint that might be important for the process giving rise to the alpha and beta halves of the 26S rRNA. Correspondence to: E. Gorab