甘油脱水酶gldC基因克隆、表达与鉴定

目的：表达及纯化甘油脱水酶γ亚基蛋白。方法：使用PCR及DNA重组技术,将甘油脱水酶7亚基基因gldC重组到含麦芽糖结合蛋白(MBP)的融合蛋白表达载体pMAL—c2x中,在大肠杆菌中进行表达。结果：转化重组质粒pMAL/gldC的大肠杆菌经IPTG诱导,SDS—PAGE分析显示表达出的MBP—gldC融合蛋白相对分子质量约66kD,与预期大小一致,并经Western blot分析证实。用直链淀粉树脂亲和层析纯化得到电泳均一的融合蛋白。结论：成功地获得甘油脱水酶γ亚基融合蛋白,以便进一步研究其生物学性能。     

MBP-gldABC融合蛋白基因的原核表达载体的构建

目的：构建可高效生产甘油脱水酶的大肠杆菌工程菌,方法：将编码甘油脱水酶的三个基因gldA、gldB、gldC,分别克隆至克隆载体pMD18-T和pSIM-T中,经测序正确后,再亚克隆至表达融合蛋白的高效表达载体pMAL-c2X上,构建成表达质粒pMAL-gldABC,并转化大肠杆菌E.coli DH5α。结果：成功地将甘油脱水酶基因gldABC以同向串联方式克隆到大肠杆菌融合表达载体pMAL-c2X中,结论：得到了含gldABC基因的MBP融合蛋白表达载体,为研究甘油脱水酶基因（gldABC）的在原核表达载体中的串联表达奠定了基础。     

重组依赖辅酶B12的甘油脱水酶基因表达系统构建

采用PCR方法从肺炎克雷伯氏杆菌基因组中分别扩增得到了编码依赖辅酶B12的甘油脱水酶α、β、γ三个亚基的基因gldA、gldB、gld将gldBC基因克隆至pSIM—T载体上,经测序正确后亚克隆至pGEX-4T-1载体中。将gldA进行T-A克隆后测定核苷酸序列正确,亚克隆至连有gldBC基因的pGEX-4T-1载体中。从而成功的构建了gldABC基因的同向串联原核融合表达载体pGEX—gldABC。     

重组人CK2β亚基的原核表达、纯化与鉴定

将构建成功的人蛋白激酶CK2β亚基cDNA的重组质粒, 转化大肠杆菌BL21 (DE3), 在IPTG诱导下表达. 表达蛋白大多数以不溶形式存在. 6L(约10.2 g)表达菌抽提得到约20 mg的可溶性表达产物, 通过P11磷酸纤维素一步层析分离, 得到6.8 mg纯化蛋白. SDS-聚丙烯酰胺凝胶电泳结果显示纯化的蛋白为一分子质量26 ku的单一蛋白带.蛋白质印迹结果证明：纯化的表达产物与抗人CK2β抗体可发生特异性免疫反应. CK2β亚基对CK2α有激活作用, 纯化的CK2α和β亚基在等摩尔混合时即可组成有最大生物活性的全酶. 实验结果有力地证明了克隆表达与纯化的重组蛋白是人蛋白激酶CK2β亚基.     

甘油脱水酶再激活酶的克隆表达及活性鉴定

运用PCR技术从克雷伯氏菌的基因组中分别扩增得到了编码甘油脱水酶再激活酶α、β两个亚基的基因gdrA、gdrB。将gdrA、gdrB克隆至pMD-18T载体上,构建克隆载体pMD-gdrAB。经测序正确后,将gdrAB亚克隆至表达载体pET-28a( )上构建表达质粒pET-28gdrAB。利用双抗生素筛选法,将pET-28gdrAB与连有甘油脱水酶基因的表达载体pET-32gldABC在大肠杆菌菌株BL21(DE3)中共表达,鉴定了甘油脱水酶再激活酶的活性。     

甘油脱水酶和二醇脱水酶β亚基生物信息学分析

依赖辅酶B12的甘油脱水酶和二醇脱水酶是同工酶。它们不仅氨基酸序列和蛋白质折叠方式非常相似,而且有着相同的催化机制。该文在同一菌株中将编码甘油脱水酶β亚基基因和编码二醇脱水酶β亚基基因片段分别克隆至T载体,从而获得两段核苷酸片段序列,将pddB序列提交Genbank数据库,获登陆号为DQ483052。同时结合PDB数据库中甘油脱水酶和二醇脱水酶的晶体结构,该文用生物信息学方法对所测序列进行了初步分析,并对两种同工酶的β亚基对脱水酶与辅酶的亲和力进行了比较。     

PSCA-HSP70融合蛋白在大肠杆菌中的表达与纯化

旨在大肠杆菌中表达PSCA-HSPT0融合蛋白,并对其进行纯化.克隆人PSCA基因及HSP70基因,构建表达PSCA-HSP融合蛋白的工程菌,优化表达及纯化条件,对重组蛋白进行纯化.结果表明,成功构建重组表达质粒PSCA-HSP,重组蛋白得到可溶性表达,优化纯化条件后获得90%以上纯度的重组蛋白.本研究成功实现了PSCA与HSP的融合表达,为下一步肿瘤疫苗的研制奠定基础.     

酮古龙酸菌细胞色素c氧化酶亚基Ⅱ的融合表达及纯化

目的:利用大肠杆菌融合表达酮古龙酸菌细胞色素c氧化酶亚基Ⅱ(CcoⅡ)与谷胱甘肽S-转移酶(GST)并纯化。方法:根据酮古龙酸菌Y25基因组序列设计引物,通过PCR扩增CcoⅡ基因,酶切后连接pGEX-KG表达载体,转化至大肠杆菌获得重组菌,经IPTG诱导表达融合蛋白GST-CcoⅡ,用谷胱甘肽-Sepharose 4B树脂亲和纯化融合蛋白,并利用Western印迹及质谱对表达蛋白进行鉴定。结果:扩增得到867 bp的CcoⅡ基因,构建了pGEX-KG-CcoⅡ融合表达载体,重组菌经0.4 mmol/L IPTG于20℃诱导16 h,SDS-PAGE分析显示有可溶性表达条带,相对分子质量约为59×103;Western印迹及质谱分析表明,利用亲和层析方法纯化到了目的蛋白。结论:表达并纯化了GST-CcoⅡ融合蛋白,为酮古龙酸菌电子传递链的研究奠定了基础。     

重组人βNGF在大肠杆菌中的可溶表达、纯化及活性鉴定

旨在大肠杆菌中可溶表达重组人神经生长因子(Recombinant humanβnerve growth factor,rhβNGF),并对表达产物进行分离纯化和生物学活性鉴定。成功扩增h NGFβ亚基基因,将其克隆入pMAL-c2X表达载体,构建了hβNGF-MBP的大肠杆菌表达体系并进行诱导表达,表达产物经纯化后以Factor Xa酶切去除麦牙糖结合蛋白(MBP),Western blot鉴定后以TF-1细胞法检测生物学活性。结果显示,pMAL-c2X-hβNGF经酶切和测序证实构建正确,25℃、180 r/min、0.5 mmol/L IPTG诱导下可溶表达hβNGF-MBP融合蛋白。hβNGF-MBP经Factor Xa酶切后可去除MBP标签,SDS-PAGE分析纯化的hβNGF位于13 k D左右,纯度可达95%。Western blot鉴定为hβNGF,结果表明,比活约为1×10~6 U/mg。在大肠杆菌中成功可溶表达hβNGF,并具有较高的生物学活性。     

重组人神经生长因子β亚基的原核优势表达及纯化

目的:在原核系统内表达SUMO融合重组人神经生长因子β亚基(h NGF-β),并对其纯化。方法:将5'端引入了羟胺切割位点的h NGF-β基因克隆到表达载体p ET-SUMO中,IPTG诱导表达后,对表达的融合蛋白SUMO-h NGF-β包涵体进行亲和纯化,然后在变性条件下加入羟胺进行裂解,利用SUMO分子伴侣的功能与h NGF-β进行共复性,最后利用阳离子交换层析纯化获得重组h NGF-β。结果:融合蛋白SUMO-h NGF-β相对分子质量为39×103,表达量占细菌总蛋白的35%;在变性条件下经羟胺切割、共复性和阳离子纯化后,相对分子质量为14×103的重组h NGF-β复性率和纯度分别为30%和80%以上,Western印迹显示其具有良好的抗原特性。结论:h NGF-β与SUMO融合可以在原核表达系统中实现优势表达。     

Entwicklungsgeschichte und Ultrastruktur von Pollenkitt und Exine bei nahe verwandten entomophilen und anemophilen Angiospermensippen derAlismataceae,Liliaceae, Juncaceae,Cyperaceae, Poaceae undAraceae

Some closely related members of the monocotyledonous familiesAlismataceae, Liliaceae, Juncaceae, Cyperaceae, Poaceae andAraceae with variable modes of pollination (insect- and wind-pollination) were studied in relation to the ultrastructure of pollenkitt and exine (amount, consistency and distribution of pollenkitt on the surface of pollen grains). The character syndromes of pollen cementing in entomophilous, anemophilous and intermediate (ambophilous or amphiphilous) monocotyledons are the same in principal as in dicotyledons. Comparing present with former results one can summarize: 1) The pollenkitt is always produced in the same manner by the anther tapetum in all angiosperm sub-classes. 2) The variable stickiness of entomophilous and anemophilous pollen always depends on the particular distribution and consistency of the pollenkitt, but not its amount on the pollen surface. 3) The mostly dry and powdery pollen of anemophilous plants always contains a variable amount of inactive pollenkitt in its exine cavities. 4) A step-by step change of the pollen cementing syndrome can be observed from entomophily towards anemophily. 5) From the omnipresence of pollenkitt in all wind-pollinated angiosperms studied one can conclude that the ancestors of anemophilous angiosperms probably have been zoophilous (i.e. entomophilous) throughout.

     

Identification and Characterization of the First Equine Parainfluenza Virus 5

正Dear Editor,Parainfluenza virus 5 (PIV5), known as canine parainfluenza virus in the veterinary field, is a negative-sense,nonsegmented, single-stranded RNA virus belonging to the Paramyxoviridae family (Chen 2018). The virus was first reported in primary monkey kidney cells in 1954 (Hsiung1972), then it has been frequently discovered in various     

Pathogenic Characterization and Full Length Genome Sequence of a Reassortant Infectious Bursal Disease Virus Newly Isolated in Pakistan

<正>Dear Editor,Infectious bursal disease (IBD) is one of the most important diseases of the poultry. The IBD virus (IBDV), a nonenveloped virus belonging to the Birnaviridae family with a genome consisting of two segments of double-stranded RNA (segments A and B), targets B lymphocytes of bursa of Fabricious leading to immunosuppression. In Pakistan,poultry farming is the second biggest industry and IBD is the second biggest disease threating the poultry sector.However, there is limited genome information of IBDV     

Similar aerosol emission rates and viral loads in upper respiratory tracts for COVID-19 patients with Delta and Omicron variant infection

Highlights
1 Aerosol emission rates of Delta or Omicron patients were similar.
2 Viral loads in upper respiratory tract of Alpha, Delta and Omicron patients were similar.
3 Viral loads in upper respiratory tract of vaccinated or unvaccinated Delta patients had no difference.     

Recombinant human interferon-α1b inhibits SARS-CoV-2 better than interferon-α2b in vitro

Highlights
1) A comprehensive evaluation method for anti-SARS-CoV-2 drugs was established based on RT-qPCR, TCID₅₀ method, and immunofluorescence.
2) A significant antiviral effect of rHuIFN-α1b was shown with EC₅₀=0.12 IU/mL in Vero cells and EC₅₀=0.52 IU/mL in Calu-3 cells, which was better than rHuIFN-α2b (EC₅₀=0.25 IU/mL in Vero cells and EC₅₀=2.48 IU/mL in Calu-3 cells).
3) rHuIFN-α1b has a good potential in the application of anti-COVID-19 therapy.     

Continued antigenic variation of highly pathogenic avian influenza A (H7N9) virus in laying hens in China, 2020–2021

Highlights
1. 13 strains of H7N9 viruses from laying hens in 2020 and 2021 were identified.
2. H7N9 viruses in China comprised at least 11 genotypes.
3. H7N9 viruses are high pathogenic in chickens, not in ducks.
4. The most H7N9 viruses cross-reacted poorly with H7-Re3 antiserum.
5. The H7-Re3 vaccine was unable to prevent H7N9 infection.