Eukaryotic expression, purification and activity characterization of human soluble DSG2 extracellular domain protein

Objective:To construct a secretory eukaryotic expression vector of DSG2 fused with the Fc region of the human IgG,to validate its expression in 293T cells,and to purify the secretory protein with biological activity.Methods:The DSG2 extracellular domain fragment gene(DSG2ex),was amplified by PCR,and was inserted into the eukaryotic expression plasmid pCMV3-IgG1 to construct the recombinant eukaryotic expression plasmid-pCMV3-DSG2ex-IgG1.The successfully constructed eukaryotic expression plasmid was transfected into 293T cells to express and secrete DSG2 extracellular domain protein.The targeted protein was purified from the cell culture supernatant by Protein A affinity chromatography and confirmed by Western Blotting and ELISA.Results:The pCMV3-DSG2ex-IgG1 eukaryotic expression plasmid was successfully constructed.The highest protein expression level was obtained with 293T cells after 96 h of transfection.The relative molecular mass of the purified product was between 100 and 130 kDa was estimated by SDS-PAGE,which was consistent with the expectation.The yield of the purified protein reached 0.8 mg/ml with a purity over 90%.The purified DSG2 extracellular domain protein with IgG1 tag was recognized by IgG monoclonal antibodies by Western blotting.Moreover,the ELISA results showed that the prepared DSG2 extracellular domain protein had significant binding activity to human type 55 adenovirus Fiber Knob protein(HAdV-55).Conclusion:A simple and efficient method for eukaryotic expression and purification of human soluble DSG2 extracellular domain protein was successfully established,and biologically active DSG2 extracellular domain protein was purified,which laid the foundation for the later study of its protein function and anti-adenovirus drugs.

Soluble expression of recomb inant cMyc,Klf4,Oct4,and Sox2 proteins in bacteria and transduction into living cells

AIM：To develop a new method to produce recombinant reprogramming proteins,c Myc,Klf4,Oct4,and Sox2,in soluble format with low cost for the generation of induced pluripotent stem cells（i PSCs）.METHODS：A short polypeptide sequence derived from the HIV trans-activator of transcription protein（TAT） and the nucleus localization signal（NLS） polypeptide were fused to the N terminus of the reprogramming proteins and they were constructed into p Cold-SUMO vector which can extremely improve the solubility of recombinant proteins.Then these vector plasmids were transformed into E.coli BL21（DE3） Chaperone competent cells for amplification.The solubility of these recombinant proteins was determined by SDS-PAGE and Coomassie brilliant blue staining.The recombinant proteins were purified by NiNTA resin and identified by Western blot.The transduction of these proteins into HEK 293 T cells were evaluated by immunofluorescence staining.RESULTS：These four reprogramming proteins could be produced in soluble format in p Cold-SUMO expression vector system with the assistance of chaperone proteins in bacteria.The proteins were purified successfully with a purity of over 70% with a relative high transduction rate into 293 cells.CONCLUSION：The results in the present study indicate the four important reprogramming proteins,c Myc,Klf4,Oct4,and Sox2,can be produced in soluble format in bacteria with low cost.Our new method thus might be expected to greatly contribute to the future study of i PSCs.

Gene Sequence, Soluble Expression and Homologous Comparison of a D-Hydantoinase fromPseudomonas putida YZ-26

A 1440bp open-reading frame encoding D-hydantoinase from Pseudomonas putida YZ-26 was cloned and sequenced（ GenBank AY387829）. The DNA fragment was inserted into Nde and BamHI sites of vector pET-3a, yielding a recombinant plasmid, pET-HDT. After being transferred into the host strain, the artificial strain, pET-HDT/ E. coli BL21 can express the D-hydantoinase as the soluble form in the Lura-Bertani medium without addition of any inducers. The activity of the enzyme toward substrate DL-hydantoin can reach 3000-4000 IU per cells from one-liter bacterial culture incubated at 30 ℃ for 10-12 h. By the comparison of amino acid sequence homology, hydrophobic residues analysis and secondary structure prediction, it was found that D-hydantoinase reported herein is quite similar to that from Pseudomonas putdia CCRC12857, and alike to that from Pseudomonas putdia DSM84 or other bacteria. A rapid and efficient purification procedure of the enzyme was performed by a three-step procedure： ammonium sulfate fractionation, phenyl Sepharose hydrophobic interaction chromatography and Sephacryl S-200 gel filtration. The molecular mass of the monomeric enzyme is 52042 Da as determined by MALDI-TOF mass spectrometry.

Chemical Chaperones Increasing Expression Level of Soluble Single-chain Fv Antibody(scFv2F3)

Glutathione peroxidase（GPX） is a sclenoenzyme that protects the biomembrane and other cellular components against oxidative damage. The selenium-containing single chain Fv fragment of monoelonal antibody 2173 （Se scFv2F3） is a kind of GPX mimic and it has a wide clinical applications because of its high activity and low antigenicity. Se-seFv2F3 is generated by the chemical modification of the single chain Fv fragment of monoelonal antibody 2F3 （ scFv2F3 ）, which can be expressed in E. coll. In this article, the effect of chemical chaperones, such as glycerol, glucose, and β-cyclodextrin added to the culture medium, on the expression of soluble scFv2F3 was investigated. The expression level was evaluated by the determination of soluble scFv2F3 contents in the whole cell lysates. The results suggest that both glycerol and β-cyclodextrin greatly increase the expression level of soluble scFv2F3, and β-cyclodextrin is found to be more effective compared with glycerol. Glucose has a slight effect on the expression level of soluble scFv2F3. This is the first example, wherein β-cyclodextrin has been used as a chemical chaperone during the cell culture to improve the expression level of recombinant proteins. In addition, chemical chaperones are found to decrease the toxic effect of IPTG on cells.

Soluble Expression,Purification and Characterization of Single-chain Fv Catalytic Antibody(sFv-2F3)

To find optimal conditions for expressing the soluble form of sFv-2F3 and to study the purification and property of its derivative Se-sFv-2F3,the preferred expression conditions were investigated by means of orthogonal design. These culture conditions included incubation temperature,inducer concentration, induction time and cell concentration. The evaluation of expression was accomplished by the analysis of whole cell lysates and the yield of soluble sFv-2F3 was calculated according to the analysis of Profinder(FTI-500,Pharmacia). The purification procedure was carried out via a two-step purification procedure consisting of ion-exchange chromatography,followed by immobilized metal affinity chromatography(IMAC). The antioxidant efficacy of Se-sFv-2F3 was demonstrated by the determination of the content of the main product of lipid peroxidation,MDA,the viability of cells and the activity of LDH. We obtained the preferred culture conditions to grow the engineered bacteria and the procedure for preparing soluble sFv-2F3 and confirmed the antioxidant efficacy of Se-sFv-2F3.

Efficient and Soluble Expression of α Protein of Clostridium perfringens and Preparation of Genetic Engineering Subunit Vaccine

[Objective] The paper was to develop genetic engineering vaccine that can express α exotoxin antigen protein efficiently without destroying its immunogenicity for preventing and controlling the diseases caused by Clostridium perfringens. [Method] Efficiently expressed soluble recombinant α protein was obtained from Escherichia coli expression system by optimizing codon,removing signal peptide,selecting sequences with better hydrophilicity and antigenicity,and optimizing expression conditions. [Result] Mice obtained higher serum antibody level when immunized by α protein,and the immune protection rates against type A,type B,type C and type D C. perfringens were 100%,90%,85% and 90%,respectively. The antibody titer of mice within 7-14 d after the third immunization reached the peak. [Conclusion]The α protein has good immunogenicity,and can be further used to develop genetic engineering subunit vaccines for preventing C. perfringens.

Cloning and Expression of the cDNA of a Murine Soluble Fas

In order to regulate the apoptosis induced by Fas FasL system, a soluble isoform of mouse Fas was cloned from thymocytes of immature mice with the primers designed according to the full length Fas cDNA sequence in the GeneBank. It was directionally inserted into the intermedium vector pUC19. DNA sequencing proved that it was consistent with the expected sequence. Then it was subcloned into the eukaryotic expression vector pCA13, which was used to construct the recombinant vector pCA13 FasC. By lipofectamine (LF2000) mediated transfection, pCA13 FasC was transfected into the 293 cells. RT PCR and Western blot indicated that the murine soluble Fas C protein was expressed in the 293 cells. Apoptosis inducing test showed that the expression of this murine Fas C could block the Fas induced apoptosis, which confirmed the biological activity of the recombinant Fas C.

Efficient and Soluble Expression of N Protein of Peste Des Petits Ruminants Virus and Development of Indirect ELISA

[ Objective] The paper was to get effective soluble N protein to establish indirect ELISA method for Peste des petits ruminants （PPR）. [ Method] Soluble N protein with high expression was obtained from Escherichia coli expression system through codon optimization and optimization of expression conditions, and indirect ELISA detection method based on N protein was further established. [ Result] The assay had no cross reaction with other sheep pathogens. The intra- and inter-batch variation coefficients were less than 9%, indicating the method had good repeatability. Furthermore, totally 480 clinical serum samples were detec- ted by the assay, and the agreement rate with commercial ELISA kit （IDVET） was 98.33%. [ Conclusion] The study laid a foundation for further development of mature PPRV antibody detection kits.

High-level expression and purification of Plutella xylostella acetylcholinesterase in Pichia pastoris and its potential application

The acetylcholinesterase 2（AChE2）cloned from Plutella xylostella was first successfully expressed in methylotrophic yeast Pichia pastoris GS115.One transformant with high-level expression of the recombinant AChE（rAChE,23.2 U mL-1in supernatant）was selected by plating on increasing concentrations of antibiotic G418 and by using a simple and specific chromogenic reaction with indoxyl acetate as a substrate.The maximum production of r ACh E reached about 11.8 mg of the enzyme protein per liter of culture.The r ACh E was first precipitated with ammonium sulfate（50%saturation）and then purified with procainamide affinity column chromatography.The enzyme was purified 12.1-fold with a yield of 22.8%and a high specific activity of 448.3 U mg-1.It was sensitive to inhibition by methamidophos and pirimicarb,the calculated 50% inhibitory concentration（IC50）values of the two pesticides were 0.357 and 0.888 mg L-1,respectively,and the calculated 70% inhibitory concentration（IC70）values were 0.521 and 0.839 mg L-1,respectively.The results suggested that it has a potential application in the detection of pesticide residues.

Construction of Lactuca sativa Plastid Transformation Vector and High-level Expression of gfp Gene in Escherichia coli

Using genomic DNA of bolting-tolerant lettuce as a template,flanking fragments of lettuce plastid rpo A gene were amplified and cloned by PCR. Targeting the sites of these two fragments,homologous recombinant fragments of exogenous gene were integrated to construct lettuce plastid expression vector p Brpo AGFP,which harbored the expression cassette Prrn-gfp-aad A-Tpsb A. The results showed that the amplified flanking fragments were 1.2 and 1.1 kb in size. After sequencing,restriction digestion,ligation and transformation,lettuce plastid expression vector containing expression cassette Prrn-gfp-aad A-Tpsb A was constructed and confirmed by SDS-PAGE electrophoresis. The results of SDS-PAGE electrophoresis indicated that gfp gene was efficiently expressed under the regulation of plasmid specific promoter Prrn and terminator Tpsb A. GFP accounted for 45. 6% of total soluble proteins; inclusion bodies accounted for 47.5 % of bacterial proteins,which reached relatively high expression levels. The construction of lettuce plastid expression vector p Brpo A-GFP laid a solid foundation for establishment of subsequent lettuce plastid transformation system and genetic improvement of lettuce using various functional genes.

Primary Purification of Co-expressed Soluble and Insoluble Alpha-interferon 2b from Recombinant E. coli

Alpha-interferon 2b (IFN 2b) was produced both in soluble and insoluble forms from recombinant E. coli. The dissolution of the expressed IFN 2b in inclusion body was carried out and it was found that the optimal condition to dissolve the expressed protein was 7 mol稬1guanidinium salt solution at pH 3.0. The resultant solution was diluted 20 times using pH 6.0 buffer to refold the protein correctly. The cation exchange column was employed to purify both refolded and soluble IFN 2b. For soluble IFN sample, high IFN 2b recovery yield (92.1%) with 91.7% purity was obtained in the eluate. However, for refolded IFN sample, only 72.7% of IFN 2b was recovered with relatively low purity (56.8%) by cation exchange chromatography. Although the expression level of insoluble IFN was higher than that of co-expressed soluble IFN in this recombinant E. coli cells, the productivity of bioactive IFN 2b was higher with soluble expressed IFN after primary purification process. Soluble expression of foreign proteins in recombinant bacteria might be an alternative strategy for efficient production of heterogeneous pro-teins due to high bioactivity and simple downstream protein purification process.

Expression,purification and identification of LBD domain of human PPARδ in E.coli

Peroxisome proliferator-activated receptors(PPARs) are ligand-activated transcription factors known to play a pivotal role in regulations of metabolism.In order to yield soluble ligand binding domain of PPARδ(PPARδLBD) for screening ligands,the cDNA was amplified using total RNA from HepG2 cells by RT-PCR.Then the enzyme-digested product was inserted downstream of the malE gene in the vector pMAL-p2x,which encoded maltose-binding protein(MBP),resulting in the expression of an MBP-PPARδLBD fusion protein.The recombinant plasmid was transformed into E.coli TB1 that was cultured shakily at 30 °C,200 r/min and induced by 0.4 mmol/L IPTG for 6 h.The cells were harvested by centrifugation and broken by sonication.The expressed fusion protein was soluble and accounted for 0.31 of the total protein in the supernatant.Western blot analysis showed that the expressed MBP-PPARδLBD could bind to anti-MBP-antibody.The MBP-PPARδLBD fusion protein of 77 kDa and the PPARδLBD protein of 34 kDa were obtained by amylose-resin affinity chromatography without or with digestion of Factor Xa.They were both homogeneity,judged by SDS-PAGE.The recombinant MBP-PPARδLBD and PPARδLBD protein with high purity is obtained,which provides the necessary material for screening and researching PPARδ ligands.

枸杞SWEET基因家族鉴定及其与可溶性糖含量关系

【目的】糖外排转运蛋白(sugars will eventually be exported transporters,SWEETs)在植物生长发育过程中发挥重要作用,解析SWEETs基因在枸杞果实发育过程中对糖积累作用,为进一步揭示SWEETs基因在枸杞果实发育过程中的作用提供参考。【方法】用生物信息学方法对枸杞SWEET基因(LbaSWEETs)进行全基因组鉴定,并用已发表的转录数据分析LbaSWEETs在果实发育时期的基因表达情况。【结果】枸杞SWEET基因家族共有37个成员,随机分布于10条染色体上,分别编码152~621个氨基酸,蛋白质分子质量为16.87~69.97 kD,等电点为4.96~9.86。亚细胞定位预测位于叶绿体或质膜,大多数含有7个跨膜螺旋。系统进化分析发现,37个LbaSWEETs蛋白可分为4个亚群,每个亚群的基因结构和保守基序组成相似。启动子元件分析表明:Lba-SWEETs基因启动子富含大量激素响应、逆境胁迫和生长发育响应元件。转录组数据和qRT-PCR分析表明:LbaSWEET9和LbaSWEET29基因表达量随果实成熟呈现显著增加。相关性分析结果表明,LbaSWEET9和LbaSWEET29基因表达量与果糖含量呈显著正相关。【结论】LbaSWEET9和LbaSWEET29基因是果糖积累的关键基因。

丹红注射液联合肝素治疗脓毒症并发急性肾损伤的效果

目的探讨丹红注射液联合肝素治疗脓毒症并发急性肾损伤的临床疗效及对血清高迁移率族蛋白B1(high mobility group box 1,HMGB1)和可溶性髓样细胞触发受体-1(soluble triggering receptor expressed on myeloid cells-1,sTREM-1)水平的影响。方法选取收治的80例脓毒症并发急性肾损伤患者作为研究对象,用随机数字表法分为2组,每组40例,2组均予以常规治疗,对照组加用低分子肝素,观察组加用低分子肝素和丹红注射液。对比2组的治疗效果,记录2组治疗前后的血清HMGB1、sTREM-1、肾功能指标及凝血功能指标等的变化。结果与治疗前比较,2组治疗后的各项生命指标、炎症指标、凝血功能指标、肾功能指标、HMGB1及sTREM-1水平均得到明显改善(P<0.05)。与对照组比较,观察组治疗后的体温、心率及急性生理学及慢性健康状况评分系统Ⅱ(acute physiology and chronic health evaluationⅡ,APACHEⅡ)评分均明显更低,血清白细胞介素-6(interleutin-6,IL-6)、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、C反应蛋白(C-reactive protein,CRP)、降钙素原(procall⁃citonin,PCT)、血肌酐(screatinine,Scr)、胱抑素C(cystatin C,CysC)、尿素氮(blood urea nitrogen,BUN)、纤维蛋白原(fibrinogen,FIB)及D-二聚体(D-dimer,D-D)的水平均更低,机械通气时间及ICU住院时间明显更短,凝血酶原时间(prothrombin time,PT)明显更长(P<0.05)。2组治疗后的存活率比较差异无统计学意义(P>0.05)。结论丹红注射液联合肝素治疗能够通过抑制sTREM-1、HMGB1的表达,减轻炎症反应,改善凝血功能,增大肾灌注,减轻肾组织损伤,从而治疗脓毒症并发急性肾损伤。

老年舒张性心力衰竭合并肌少症患者可溶性生长刺激表达基因2蛋白、肌红蛋白、白细胞介素-6水平与心功能的相关性

目的 探讨老年舒张性心力衰竭(DHF)合并肌少症患者外周血可溶性生长刺激表达基因2蛋白(sST2)、肌红蛋白(Myo)、白细胞介素-6(IL-6)水平与心功能的相关性。方法 将122例DHF患者根据有无肌少症分为DHF合并肌少症组60例和DHF组62例,另将健康体检者58例、单纯肌少症患者60例分别纳入对照组、单纯肌少症组,检测各组外周血sST2、Myo、IL-6水平和心功能指标[左室射血分数(LVEF)、心排血量(CO)、心率(HR)、每搏输出量(SV)和心脏指数(CI)]。采用Pearson相关分析法分析sST2、Myo、IL-6与各心功能指标的相关性。绘制受试者工作特征(ROC)曲线,分析sST2、Myo、IL-6单独及联合诊断DHF合并肌少症的效能。结果 与对照组、单纯肌少症组相比,DHF组、DHF合并肌少症组sST2、Myo、IL-6水平和HR均升高,LVEF、CO、SV和CI均降低,差异有统计学意义(P<0.05);与DHF组相比,DHF合并肌少症组sST2、Myo、IL-6水平和HR均升高,LVEF、CO、SV和CI均降低,差异有统计学意义(P<0.05)。sST2、Myo、IL-6均分别与LVEF、CO、SV、CI呈负相关(P<0.001),均与HR呈正相关(P<0.001);sST2、Myo、IL-6、LVEF、SV是DHF合并肌少症的独立影响因素(P<0.05);sST2、Myo、IL-6联合诊断DHF合并肌少症的曲线下面积为0.936,诊断效能优于三者单独检测。结论 老年DHF合并肌少症患者外周血sST2、Myo、IL-6水平显著升高,且sST2、Myo、IL-6均与心功能指标显著相关,三者联合检测对DHF合并肌少症的诊断效能较高。

血清IL-2、sST2表达与特发性膜性肾病免疫抑制剂治疗反应性的相关性

目的探讨特发性膜性肾病患者血清白介素-2(IL-2)、可溶性生长刺激表达基因2蛋白(sST2)表达水平与免疫抑制剂治疗反应性的相关性。方法选取2020年1月至2022年10月于医院接受免疫抑制剂治疗的135例特发性膜性肾病患者,于入院时检测患者血清IL-2、sST2,并于治疗完成后测定24 h尿蛋白定量,依据患者治疗反应性分为缓解组与未缓解组。对比两组患者一般资料及入院时血清IL-2、sST2水平,采用点二列相关性分析血清IL-2、sST2水平与特发性膜性肾病免疫抑制剂治疗反应性的关系,并绘制受试者工作特征(ROC)曲线评估血清IL-2、sST2水平预测特发性膜性肾病免疫抑制剂治疗反应性的价值。结果135例特发性膜性肾病患者中共有132例完成规律治疗,经免疫抑制剂治疗6个月后,101例患者疾病缓解,纳入缓解组,其余31例患者纳入未缓解组。未缓解组年龄、入院时肾功能分级、疾病分期、血清IL-2、sST2水平均高于缓解组,差异有统计学意义(P<0.05);点二列相关性分析显示,血清IL-2、sST2水平与特发性膜性肾病免疫抑制剂治疗反应性不良风险呈正相关(r 1=0.428,P 1<0.001;r 2=0.344,P 2<0.001);绘制ROC曲线,结果显示,血清IL-2、sST2预测特发性膜性肾病免疫抑制剂治疗反应性不良的曲线下面积均>0.7,具有一定预测价值,且联合预测价值更高。结论血清IL-2、sST2表达水平与特发性膜性肾病患者免疫抑制剂治疗反应性密切相关,二者表达水平越高,治疗反应性越差,且联合检测可作为预测特发性膜性肾病患者免疫抑制剂治疗反应性的敏感指标。

白喉毒素突变体CRM197在大肠杆菌中优化表达及人群血清抗体水平分析

目的 构建白喉毒素突变体CRM197的原核表达载体,在大肠杆菌中表达CRM197蛋白。分析健康人群血清中的CRM197抗体水平。方法 对CRM197基因进行大肠杆菌密码子优化并克隆至pET28a(+)中,经表达条件优化后采用镍柱亲和层析和离子交换层析对重组蛋白CRM197进行纯化,对纯化的CRM197进行Western blot鉴定。最后,经纯化后的CRM197蛋白与健康人群血清进行反应,检测人群血清中针对该蛋白的抗体水平。结果 CRM197蛋白进行了密码子及表达条件优化后,获得了可溶性表达蛋白。经镍柱亲和层析和离子交换层析后其纯度可达95%以上,Western Blot鉴定后能得到单一的特异性条带。经纯化的CRM197蛋白与不同年龄阶段健康人群血清均发生不同程度的抗原抗体反应。结论 利用本研究的策略,CRM197得到高效的可溶性表达,但人群血清中含有高水平的CRM197抗体,这些抗体的存在可能会影响到应用CRM197结合多糖作为抗原进行特异免疫效果的评价以及作为诊断抗原的应用。

热纤梭菌耐热葡聚糖外切酶的克隆表达及酶学特性分析

研究旨在开发耐热纤维素酶,以提高畜禽对纤维素饲料的利用率和养殖经济效益。从蛋白质信息数据库UniProt中获取热纤梭菌(Clostridium thermocellum)葡聚糖外切酶CelS的序列信息,经信号肽删除和密码子优化,利用大肠杆菌表达系统,实现了CelS的过量表达。通过自诱导表达条件优化,有效提高了CelS可溶表达的比例。将胞内上清液通过镍离子亲和层析和热处理分离纯化获得可溶CelS,将胞内沉淀通过洗涤、变性和复性后获得包涵体复性CelS。酶学特性检测发现,两种表达形式CelS的酶活力无显著性差异,最适反应温度均为70℃,最适反应pH值为5.5~6.0,对无定形纤维素(PASC)的活性最高,对结晶纤维素(Avicel)和玉米秸秆(CS)次之。CelS与海栖热袍菌(Thermotoga maritima)葡聚糖内切酶EG12B协同作用水解CS,比单独水解酶活力提高了81.8%。试验结果表明,重组CelS表现出优越的耐热性能和纤维素水解活性,为进一步开发酶制剂提供了有力支持,有利于提高纤维素饲料的利用率。

重组牛乳过敏原β-乳球蛋白的可溶性表达及其特性分析

目的实现β-乳球蛋白(β-lactoglobulin,BLG)的可溶性表达,并比较重组BLG和天然BLG在结构和免疫原性两个方面的差异性。方法将pET-30a-BLG重组质粒转入大肠杆菌BL21(DE3)感受态细胞中,用异丙基-β-D-硫代半乳糖苷(isopropyl-β-D-thiogalactopyranoside,IPTG)进行体外诱导表达,并对表达条件进行优化,采用十二烷基硫酸钠-聚丙烯酰胺电泳方法分析重组蛋白的可溶性。利用荧光光谱和圆二色谱比较重组BLG空间结构变化,同时采用竞争抑制酶联免疫吸附实验评价重组BLG致敏性能力。结果在Tris-HCl溶液体系中重组BLG的可溶性表达可达到50%以上,可溶性表达的最优条件为:在Tris-HCl溶液体系中,诱导温度为37℃,IPTG终浓度1.0 mmol/L,诱导4 h。重组BLG表面疏水性下降,可溶性重组BLG的α-螺旋结构减少,经除盐处理后致敏性下降,包涵体重组BLG无规则卷曲结构减少、致敏性上升。结论本研究成功从上清液中纯化得到可溶性重组BLG,且可溶性表达获得的BLG空间结构更接近天然蛋白。

葡萄TST基因家族鉴定及表达分析

【目的】液泡膜糖转运蛋白(TST或者TMT)是植物发育过程中发挥重要作用的一种糖转运蛋白。研究旨为探究TST基因家族在葡萄生长发育中的作用,并进一步为阐明TST基因功能提供坚实的基础。【方法】通过同源分析法从葡萄基因组中鉴定出13个TST基因,对基因的结构和编码蛋白质进行生物信息学分析;用qRT-PCR技术分析‘鄞红’葡萄发育过程中不同组织的TST表达水平,并对不同时期葡萄果肉中可溶性糖含量进行相关性分析。【结果】TST基因家族分布在6条染色体上,存在3对片段重复和3对串联重复基因;根据系统发育将其分为3个亚家族,各亚家族成员结构相似;TST基因含有大量与激素、光和胁迫响应相关的顺式作用元件;TST家族蛋白质由α-螺旋和无规则卷曲组成,各亚族蛋白模型相似;VvTST在不同组织中均有表达,并存在时空表达特异性;相关性分析发现,葡萄果肉中4个VvTST基因(VIT_18s0001g12560、VIT_18s0122g00850、VIT_04s0023g01860和VIT_03s0038g03940)的表达水平与其中可溶性糖的积累呈现相似变化趋势。【结论】VvTST可能对葡萄果肉中可溶性糖积累起到至关重要的作用。