解淀粉芽孢杆菌YN-1抑菌蛋白TasA基因的克隆及原核表达

本文以解淀粉芽孢杆菌YN-1菌株为研究对象,利用PCR方法从基因组DNA中扩增出编码TasA抑菌基因的全长DNA,并构建pGEX-4T2/TasA原核表达载体,获得TasA-GST融合表达的抑菌蛋白。测序结果表明,解淀粉芽孢杆菌YN-1TasA基因核苷酸序列全长为786bp(GenBank登录号:EU131674),编码261个氨基酸残基;同源性分析表明,它与解淀粉芽孢杆菌B.amyloliquefaciens FZB42(YP_001421886)序列的同源性最高,达98%,预测蛋白分子量为31kD。SDS-PAGE分析表明,TasA基因能在大肠杆菌BL21中表达,Western印迹分析pGEX-4T2/TasA原核表达载体,检测到约57kD的TasA-GST融合外源蛋白,与预测的融合蛋白分子量大小相符。表达产物细胞裂解液上清经Ni柱层析,表明浓度为500mmol/L咪唑洗脱缓冲液时获得较高浓度和纯度的纯化蛋白。进一步抑菌活性分析表明表达产物对黄瓜灰霉病病原菌检测平板上显示出较好的抑菌活性。本研究结果将为今后深入研究TasA抑菌蛋白基因以及抗病转基因工程提供了参考。     

不同处理方法对虾过敏蛋白分子量及抗原性的影响

本文研究不同处理方法对虾过敏蛋白分子量及抗原性的影响。利用酶解、超高压、微波3种方法处理虾过敏蛋白,利用SDS-PAGE对虾过敏蛋白分子量大小进行测定,用OPA法对各种酶解条件下蛋白质的水解度进行测定,间接竞争ELISA法测定各种处理后过敏蛋白抗原性的变化。结果表明蛋白酶处理后虾过敏蛋白特征条带消失,而经超高压和微波处理的分子量大小没有变化;间接竞争ELISA检测表明三种处理均会导致虾过敏蛋白致敏性不同程度降低或消失。     

仿刺参ACE抑制肽的纯化及其分子量的测定

利用仿刺参体壁制备具有抑制血管紧张素转移酶(ACE)活性的多肽,为进一步研究ACE抑制肽提供理论基础。本研究采用动物蛋白水解酶酶解仿刺参体壁,得到具有ACE抑制活性的酶解液;将酶解液粗品通过截留分子量5KDa的超滤系统,将活性部分继续用Sephadex G15分离纯化,采用HPLC法测定各组分的ACE抑制活性,同时利用RP-HPLC测定其相对分子质量分布。结果可知:仿刺参活性较高的ACE抑制肽主要由二肽到五肽组成,且分子量越小,抑制活性越高,得到了具有较高ACE抑制活性且组成比较单一的组分d,其IC50为439μg·mL-1。     

苦瓜Mp-21.6蛋白的分离及生化特性

为研究苦瓜蛋白的有效分离技术与抑菌活性,本实验采用(NH4)2SO4分级分离油苦瓜(Momordic a charantiaL.)蛋白质,将SephadexG-100凝胶柱在DEAE-52阴离子交换柱色谱工作站上串联,分步聚集纯化出苦瓜蛋白Mp-21.6。SDS-PAGE凝胶电泳显示单一蛋白条带;经生化分析鉴定,Mp-21.6(Momordica protein of 21.6kD)蛋白的分子量为21.6kD,等电点pI=9.6;双向还原SDS-PAGE确定苦瓜蛋白Mp-21.6仅存在链内二硫健;Aacomplement软件的基于氨基酸组成比较发现,Mp-21.6类似于RNMC_MOMCH[Ribonuclease MC (RNase MC,EC=3.1.27.1)]。抑菌试验显示Mp-21.6对大肠杆菌(Escherichia coli)、金黄色葡萄球菌(Staphylococcus aureus)、沙门氏菌(Salmonellab sp)、枯草芽胞杆菌(Bacillus sabtilis)、啤酒酵母(Saccharomyces cerevisiae)和黄曲霉(Aspergillus flavus)产生明显...     

鸡白介素-2基因在大肠杆菌中表达及多克隆抗体制备*

将编码鸡(Gallus gallus)白细胞介素-2(interleukin-2,IL-2)蛋白的基因亚克隆到大肠杆菌(Eschetichia coli)原核表达载体pPROEX^HT中，构建重组质粒并进行确证性序列测定。然后将重组质粒转化大肠杆菌DH5α并用IFTG于37℃诱导培养。SDS-PAGE和Western blot分析显示，表达的鸡IL-2融合蛋白分子量约为19kD。融合蛋白经薄层扫描发现目的蛋白表达量约占菌体蛋白的30％。包涵体被6mol／L，盐酸胍裂解后，通过镍离子亲和树脂进行了纯化。用所获得的重组鸡IL-2融合蛋白及其纯化产物免疫家兔，制备兔抗鸡IL-2多克隆抗血清，并用琼脂扩散实验对多克隆抗血清进行鉴定，表明其与纯化的重组鸡IL-2蛋白具有良好的反应性，而且该反应是特异的。     

中国传统细菌型豆豉溶栓酶的提取纯化技术研究(简报)

为深入开发利用中国传统发酵细菌型豆豉溶栓酶,对豆豉溶栓酶分离纯化工艺进行了研究探讨,确认了最佳的纯化条件:首先用饱和度75%的硫酸铵沉淀豆豉溶栓酶;然后利用DEAE-Sepharose FF (Diethylaminoethyl Sepharose Fast Flow,二乙基氨基琼脂糖快速凝胶)阴离子交换柱进行层析,优化的层析条件为用10 mmol/L Tris(Trishydroxymethylaminomen,三羟甲基氨基甲烷)-HCI (pH 8.7)作为上样缓冲液,采用线性梯度洗脱,洗脱液为10mmol/L Tris-HCI(pH 8.7)和0.6mol/LNaCl,洗脱流速为1 mL/min;最后经过Sephadex G-75(葡聚糖凝胶G-75)凝胶过滤,得到纯化倍数为11.29倍的豆豉溶栓酶.利用SDS-PAGE (Sodium dodecyl sulphate-polylamide gel electroresis,十二烷基硫酸钠-聚丙烯酰胺凝胶电泳) 显示经纯化后的酶无杂蛋白,初步鉴定其分子量接近31 ku.     

Fusion Expression and Purification of Marek’s disease virus Tegument Protein VP16 and Its Antibody Preparation

马立克氏病病毒(Marek’s disease virus ,MDV)UL48基因编码的蛋白与单纯疱疹病毒1型(Herpes simplex virus ,HSV)衣壳蛋白VP16为同源物，将其克隆入pET-32a载体中，获得pET-VP16, 转化Escherichia coli BL21感受态细胞，经IPTG诱导表达，SDS-PAGE电泳显示:融合蛋白获得可溶性表达，表达蛋白的相对分子量约为67 kD；将表达的融合蛋白过His·Bind柱，获得纯化的蛋白，将该蛋白免疫家兔，制备多克隆抗体。通过间接ELISA和Western blot鉴定制备的多克隆抗体的效价和特异性，结果表明，该抗体具有较高的特异性，间接ELISA效价大于2×10－5。     

重组MBP-GnRH6融合蛋白的表达与鉴定

将人工合成促性腺激素释放激素(GnRH)六聚体基因片段连接至pMAL-c4x载体,获得的pMAL-c4x-GnRH6重组质粒转化到大肠杆菌TB1中进行表达.采用SDS-PAGE,Western blot进行鉴定,并通过动物实验对表达产物的免疫原性进行检测.获得了分子量大小约为50 kD的麦芽糖结合蛋白-GnRH六聚体(MBP-GnRH6),且融合蛋白与GnRH抗体的反应性较好,并能使睾丸萎缩、变性.表明重组MBP-GnRH6融合蛋白具有较好的反应原性和免疫原性,可用于小鼠的免疫去势.     

茶树黄酮醇合成酶基因的克隆与原核表达

本研究采用EST测序技术和RT—PCR技术,获得了一个茶树茶多酚代谢中的重要基因——黄酮醇合成酶（FLS）基因,在GenBank登录（GenBank accession No．EF205150）,其序列全长1317bp,其中开放阅读框长996bp,编码331个氨基酸,3]端有一个明显的多聚腺苷酸加尾信号,推测的蛋白分子量约为37．5kD,理论等电点为5．80。序列分析表明它与葡萄FLS基因序列的亲缘关系比较近。将该基因重组到表达载体pET-32a（＋）中进行原核表达,经IPTG诱导、SDS-PAGE检测,结果表明茶树黄酮醇合成酶基因能在大肠杆菌BL21中表达,电泳检测到一条大约61kD的外源蛋白,与预测的融合蛋白分子量相符。用Ni-NTA亲和层析柱对融合蛋白进行纯化,得到了纯度在90%以上的纯化蛋白,为进一步研究PET-FLS融合蛋白的活性及功能奠定了基础。     

棉铃虫中肠.Bt 毒素受体蛋白(APN )的分离与纯化

用Mg／EGTA方法，以不同的速度进行离心，成功地分离了棉铃虫(Bacillus thuringiensis)中肠刷状缘膜囊泡（BBMVO，BBMV中保留了大部分氨肽酶N(aminopeptidase N，APN)活性，BBMV中APN的特异活性较粗匀浆液相比提高了3．29倍；CHAPS3-[(3-chlor-amidopropyl)dimethylammonio]-1-propane sulphonate可以增加BBMV的溶解，加入1％(CHAPS)之后APN特异活性增加至5．21倍；利用磷酸酰肌醇特异性的磷脂酶(PI-PLC)能将APN从膜上解离下来，BBMV经PI-PLC处理后，SDS-PAGE电泳结果仅有120kD的1条带；Mono-Q和FPLC结合的方法可以纯化部分APN，SDS-PAGE电泳结果显示，经Mono-Q柱处理后的BBMV中分子量约100kD的蛋白条带加粗；棉铃虫3个抗性品系与敏感品系相比，APN特异活性差异不显著，说明棉铃虫对Bt产生抗性与APN活性的变化无关，抗性的产生可能与Bt毒素和APN结合能力的改变有关。     

Immunological characterization of recombinant soy protein allergen produced by Escherichia coli expression system

To elucidate the molecular mechanism of the allergenicity of soybean P34 protein recognized as the most allergenic protein in soybean, the protein was expressed in Escherichia coli transformed with a plasmid carrying P34 cDNA. SDS-PAGE pattern showed that the molecular weight of the recombinant P34 was approximately 2 kDa less than that of the native soybean P34. The difference in the molecular mass between these two proteins could be due to the native P34 in soybean being glycosylated at position Asn(170), whereas the recombinant protein generated in E. coli lacks this post-translational modification. Immunoblot analysis showed that both soybean and recombinant P34 proteins cross-reacted not only with polyclonal and monoclonal antibodies produced against P34 and crude soybean protein but also with patients' sera. The results suggest that the recombinant P34 is immunologically reactive, indicating that both proteins have similar epitope structures. Thus, the recombinant P34 produced by the E. coli expression system can be used as a standard allergen for molecular design to reduce the allergenic structure.     

Characterization of the soluble allergenic proteins of cashew nut (Anacardium occidentale L.)

The allergens associated with cashew food allergy have not been well-characterized. We sought to identify the major allergens in cashew nut by performing IgE immunoblots to dissociated and reduced or nonreduced cashew protein extracts, followed by sequencing of the peptides of interest. Sera from 15 subjects with life-threatening reactions to cashews and 8 subjects who tolerate cashews but have life-threatening reactions to other tree nuts were compared. An aqueous cashew protein extract containing albumin/globulin was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subjected to IgE immunoblotting using patient sera. Selected IgE reactive bands were subjected to N-terminal amino acid sequencing. Each of the 15 sera from cashew-allergic subjects showed IgE binding to the cashew protein extract. The dominant IgE-binding antigens in the reduced preparations included peptides in the 31-35 kD range, consistent with the large subunits of the major storage 13S globulin (legumin-like protein). Low-molecular-weight polypeptides of the 2S albumin family, with similarity to the major walnut allergen Jug r 1, also bound IgE. The sera from eight patients who tolerate cashew but displayed allergies to other tree nuts showed only minimal or no IgE binding to cashew. Cashew food allergy is associated with the presence of IgE directed against the major seed storage proteins in cashew, including the 13S globulin (legumin group) and 2S albumins, both of which represent major allergen classes in several plant seeds. Thus, the legumin-group proteins and 2S albumins are again identified as major food allergens, which will help further research into seed protein allergenicity.     

Purification of hydroperoxide lyase from green bell pepper (Capsicum annuum L.) fruits for the generation of C6-aldehydes in vitro

The aim of this work was to compare the efficiency of different extracts of hydroperoxide lyase from green bell peppers in producing aldehydes: a crude extract, a chloroplastic fraction, and a purified enzyme were investigated. From a crude extract, the HPO lyase was purified by ion-exchange chromatography with a 22.3-fold increase in purification factor. Analysis by SDS-PAGE electrophoresis under denaturating conditions showed only one protein with a molecular weight of 55 kDa, whereas size-exclusion chromatography indicated a molecular weight of 170 kDa. A maximum of 7500 mg of aldehydes per g of protein was obtained with the purified enzyme within 20 min of bioconversion compared to 392 and 88 mg of aldehydes per g of protein within 50 and 60 min, respectively, for the chloroplast fraction and the crude extract.     

Seed protein electrophoretic characterization of cowpea (Vigna unguiculata) germplasm from IITA gene bank

Summary The total seed protein, globulin and albumin fractions of 20 cowpea (V. unguiculata) accessions from IITA gene bank were investigated by SDS-polyacrylamide gel electrophoresis. Total seed protein extracts were prepared from the defatted meal by homogenisation and centrifugation in Tris-glycine buffer. The globulins and albumins were obtained from the total protein extract by exhaustive dialysis against sodium acetate buffer. Two main total seed protein electrophoretic patterns were observed with respect to the 39 and 20 kD subunits, which were present in six out of the twenty accessions analysed. While there was no correlation between seed colour and total seed protein banding pattern, the six insect-resistant cultivars were characterized by the presence of the 39 and 20 kD subunits. The globulins were the predominant class of the total seed proteins and consisted mainly of 64, 58, 56 and 14 kD subunits which make up CP1 and CP2, the major globulins. The albumins in all accessions were a heterogeneous protein fraction consisting of both high and low molecular weight subunits. It was suggested that the insect-resistant cultivars may be genetically related and that the 39 and 20 kD subunits may be involved in the insect resistance mechanism.     

Characterization of a calcium-soluble protein fraction from yellow mustard (Sinapis alba) seed meal with potential application as an additive to calcium-rich drinks

A calcium-soluble protein isolate (CSPI) was prepared from the supernatant obtained after addition of 0.75 M calcium chloride to a pH 5.0 aqueous extract of yellow mustard (Sinapis alba) seed meal. Total amino acid analysis showed that the CSPI has significantly higher (p < 0.05) contents of glutamic acid + glutamine, cysteine, and proline when compared to the precipitated, calcium-insoluble proteins. Peptide mass fingerprinting of tryptic peptides of the major polypeptides by mass spectrometry indicated that the CSPI is composed mainly of cruciferin proteins with a contribution from napins (the major allergenic proteins of S. alba). The S. alba CSPI had significantly higher (p < 0.05) protein solubility and emulsion formation ability in the presence of 0.75 M calcium chloride when compared to similar isolates prepared from Brassica juncea (brown mustard) and soybean seed meals. We suggest that the S. alba CSPI could be used to prepare calcium-fortified high protein liquid products. However, the presence of allergenic proteins in this extract may limit its widespread food use.     

Differential detection of shrimp and crab for food labeling using polymerase chain reaction

Shrimp and crab are well-known as allergenic ingredients. According to Japanese food allergy labeling regulations, shrimp species (including prawns, crayfishes, and lobsters) and crab species must be differentially declared when ≥10 ppm (total protein) of an allergenic ingredient is present. However, the commercial ELISA tests for the detection of crustacean proteins cannot differentiate between shrimp and crab. Therefore, two methods were developed to discriminate shrimp and crab: a shrimp-PCR method with postamplification digestion and a crab-PCR method that specifically amplifies a fragment of the 16S rRNA gene. The sensitivity and specificity of both PCR methods were verified by experiments using DNA extracted from 15 shrimp species, 13 crab species, krill, mysid, mantis shrimp, other food samples (cephalopod, shellfish, and fish), incurred foods, and commercial food products. Both PCR methods could detect 5 pg of DNA extracted from target species and 50 ng of genomic DNA extracted from incurred foods containing 10 ppm (μg/g) total protein of shrimp or crab. The two PCR methods were considered to be specific enough to separately detect species belonging to shrimp and crab. Although false-positive and false-negative results were obtained from some nontarget crustacean species, the proposed PCR methods, when used in conjunction with ELISA tests, would be a useful tool for confirmation of the validity of food allergy labeling and management of processed food safety for allergic patients.     

Presence of allergenic proteins in different peach (Prunus persica) cultivars and dependence of their content on fruit ripening

It has been reported that various cultivars of fruits and vegetables may present a different pattern for the contained allergens. Here, we report on the different content in allergenic proteins for different peach (Prunus persica) cultivars, sampled during two consecutive harvest seasons. Fruits from six cultivars of peaches were harvested fully ripe, and the proteins extracted from whole or chemically peeled fruits were analyzed by SDS-PAGE and immunoblotting. All the protein extracts from whole fruit contained a 9 kDa protein. This protein proved to be absent in the extracts taken from chemically peeled fruit. In four cultivars, this protein corresponds to the allergen Pru p3, a lipid transfer protein that causes the oral allergy syndrome (OAS) in sensitized people. In the following year, fruits from four of the six cultivars of peaches studied previously were harvested at different times, at one and two weeks before the commercial ripening time and when fully ripe, to ascertain whether the presence of the 9 kDa allergen might be related to the ripening process. Two cultivars out of four produced an intense allergenic band corresponding to a 9 kDa protein already two weeks before the commercial ripening date, while the others showed a progressive increment of the 9 kDa allergen during ripening.     

Purification, cloning, and immunological characterization of arginine kinase, a novel allergen of Octopus fangsiao

Arginine kinase (AK) is an important enzyme participating in energy metabolism in invertebrates, but, to date, there have been no reports that AK from octopus is an allergen. In this study, octopus AK was purified, and its molecular biological, immunological, and physicochemical characterizations were analyzed. The results showed that octopus AK was purified and confirmed by mass spectrometry for the first time, and its molecular mass was 38 kDa. The full-length gene sequence of octopus AK encompassed 1209 bp and was predicted to encode a protein with 348 amino acid residues. The homology of octopus AK and crustacean AK was about 54%, but the similarity between their three-dimensional structures was high. Octopus AK could react with mouse anti-shrimp AK and rabbit anti-crab AK polyclonal antibody singly. Octopus AK could also react with specific IgE of the sera from octopus-allergic patients effectively, whereas crab AK could inhibit the reaction between them. Finally, the IgE-binding activity of octopus AK could be reduced in the processes of thermal or acid-alkali treatment. In summary, AK was identified as a novel allergen in octopus, which had a sensitizing ability similar to that of crustacean AK. This is significant in allergy diagnosis and the treatment of octopus-allergic disorders.     

棉花黄萎病拮抗细菌DS45-2菌株抗菌蛋白的分离纯化

枯草芽孢杆菌(Bacillus subtilis) DS45-2菌株产生的抗菌蛋白对棉花黄萎病有较强的抗性。用NB培养基摇床振荡培养DS45-2菌株(30℃,180r/min,48h),发酵液经硫酸铵盐析得到抗菌蛋白。经分析,抗菌蛋白对热稳定;对胃蛋白酶、胰蛋白酶均不敏感,对蛋白酶K部分敏感;在碱性条件下稳定,酸性条件下抑菌活性减弱。抗菌蛋白经DEAE Sepharose Fast Flow阴离子交换层析和反相层析后,分离纯化出一个抗菌蛋白0组分,经SDS-PAGE检测分子.质量约为23 kDa。     

Vigna vexillata (L.) A. Rich. seed proteins: heterogeneity in subunits of globulin fraction

Forty-six accessions of Vigna vexillata from Africa, America and Australia were screened for variability in globulin protein fraction by using SDS-PAGE under reducing and non-reducing conditions. The globulin fraction was composed of several bands clustering in two size fractions, both of which were polymorphic. The high molecular weight fraction was constituted by one or two subunits, while the fraction of lower molecular weight exhibited from two to four bands. Two single types were found within the collection. Due to the observed polymorphism only the subunit at 52 kD was species-specific. Southeastern Africa proved to be the region with the highest diversity for Vigna vexillata. Different frequencies of the globulin patterns were observed between the African and American groups of accessions. No significant differences were found comparing the distribution of the globulin types between the varieties vexillata and angustifolia.