抗果蝇MRJ蛋白单克隆抗体的制备和鉴定

制备抗果蝇MRJ蛋白单克隆抗体可用于研究果蝇mrj的生物学功能,使用IPTG诱导重组质粒pET28a-mrj在大肠杆菌Rosetta中表达,重组蛋白经过Ni-IDA凝胶柱亲和纯化后免疫BALB/c小鼠。然后取免疫好的小鼠的脾脏细胞与小鼠骨髓瘤细胞SP2/0融合,经克隆和筛选获得了能分泌抗果蝇MRJ蛋白单克隆抗体的杂交瘤细胞株。腹水制备后获得单克隆抗体,通过ELISA和Western Blot对所获得的抗体进行鉴定,结果表明所制备单克隆抗体能够特异性结合于原核及真核细胞表达的MRJ蛋白,可用于研究mrj基因的生物学功能。     

布鲁菌bp26基因的原核表达及间接ELISA方法的临床初步应用

构建诊断人布病的优势抗原BP26原核表达载体,并建立以该蛋白作为包被抗原的间接ELISA方法。人工合成bp26基因片段,构建原核表达载体pET30a-bp26,并转化入大肠埃希菌BL21(DE3)中,IPTG诱导表达,Ni柱亲和纯化BP26重组蛋白,间接ELISA检测其抗原活性,并优化间接ELISA方法的各个参数,检测临床大量样本,对该方法性能进行评价。PCR鉴定及DNA测序证实pET30a-bp26重组质粒构建成功,并在0.8 mmol/L IPTG 20 ℃过夜诱导的条件下大量表达,经亲和纯化获得高纯度的可溶性BP26重组蛋白,间接ELISA结果显示该重组蛋白具有抗原活性,优化参数后,该方法的敏感度为70.00%,特异性为94.74%。成功获得高抗原活性的原核表达可溶性BP26重组蛋白,并建立用于临床特异性诊断人类布病的间接ELISA方法。     

黑胸大蠊浓核病毒磷脂酶A2功能区的克隆及其表达

通过RT-PCR扩增获得PfDNV结构蛋白基因VP1含磷脂酶A2(PLA2)功能区片段,将其连接到pMD18-T载体上并亚克隆到原核表达载体pET28a和pET26b,构建阅读框架正确的重组表达载体pET28a-PLA和pET26b-PLA,转化大肠杆菌BL21-codonplus(DE3)-RIL,经IPTG诱导,SDS-PAGE显示得到了目的融合蛋白,以抗组氨酸的单克隆抗体对经Ni-NTA亲和层析柱纯化的目的蛋白进行了westernblot鉴定,结果表明成功表达PfDNV结构蛋白PLA2,对于研究该酶的生物学特性及其在病毒对细胞侵染过程中的功能奠定了基础。     

黑胸大蠊浓核病毒磷脂酶A2功能区的克隆及其表达

通过RT-PCR扩增获得PfDNV结构蛋白基因VP1含磷脂酶A2(PL A2)功能区片段,将其连接到pMD18-T载体上并亚克隆到原核表达载体pET28a和pET26b,构建阅读框架正确的重组表达载体pET28a-PLA和pET26b-PLA,转化大肠杆菌BL21-codonplus(DE3)-RIL,经IPTG诱导,SDS-PAGE显示得到了目的融合蛋白,以抗组氨酸的单克隆抗体对经Ni-NTA亲和层析柱纯化的目的蛋白进行了western blot鉴定,结果表明成功表达PfDNV结构蛋白PLA2,对于研究该酶的生物学特性及其在病毒对细胞侵染过程中的功能奠定了基础.     

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。     

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。     

人肿瘤坏死因子α抑制肽-抗炎酸性尾巴融合蛋白的原核表达及纯化

目的:表达和纯化人肿瘤坏死因子α抑制肽-抗炎酸性尾巴融合蛋白。方法:利用PCR搭接方法及基因合成方法获得目的基因,插入带有6×His标签的原核高效可溶性表达载体pET32a中,构建重组表达质粒pET32a-T9-ac-9,将重组表达质粒转化大肠杆菌BL21(DE3),经IPTG诱导目的基因表达;对融合蛋白进行Ni2+金属螯合柱纯化。结果:构建的重组表达质粒经PCR、内切酶鉴定及基因序列测定证实;目的蛋白在大肠杆菌中获得表达,SDS-PAGE显示相对分子质量为22.917×103;对表达产物进行了亲和层析纯化,从上清中获得了纯度较高的人肿瘤坏死因子α抑制肽-抗炎酸性尾巴融合蛋白。结论:获得了可溶性的人肿瘤坏死因子α抑制肽-抗炎酸性尾巴融合蛋白,为其生物学功能研究奠定了基础。     

大肠杆菌ybfE基因的三种原核质粒表达水平的对比及蛋白纯化

[目的]构建大肠杆菌功能未知基因ybf E的pET16b、pET32a和p GEX-4T-1三种原核表达系统,通过对比表达水平筛选出最优表达体系,并纯化表达的可溶性Ybf E融合蛋白。[方法]使用pET16b、pET32a和p GEX-4T-1表达质粒构建pET16b-ybf E、pET32a-ybf E和p GEX-4T-1-ybf E原核表达载体,分别转化大肠杆菌BL21,IPTG诱导表达Ybf E融合蛋白,对三种表达系统的表达水平进行对比,并对pET16b-ybf E和pGEX-4T-1-ybf E表达体系的裂菌上清中的可溶性Ybf E融合蛋白液分别使用镍柱和GST蛋白纯化柱纯化。[结果]构建了pET16b-ybf E、pET32a-ybf E和p GEX-4T-1-ybf E原核表达体系,并使用IPTG诱导表达Ybf E融合蛋白。ybf E在p GEX-4T-1载体内的表达水平最高,接下来依次为pET16b和pET32a。pET16b-ybf E和p GEX-4T-1-ybf E表达的可溶性Ybf E融合蛋白纯化后浓度分别为86μg/m L和724μg/m L。[结论]成功构建了ybf E基因的三种原核表达系统,筛选出最佳表达体系,可溶性Ybf E融合蛋白得到纯化。     

大肠杆菌ybfE基因的三种原核质粒表达水平的对比及蛋白纯化北大核心

[目的]构建大肠杆菌功能未知基因ybf E的pET16b、pET32a和p GEX-4T-1三种原核表达系统,通过对比表达水平筛选出最优表达体系,并纯化表达的可溶性Ybf E融合蛋白。[方法]使用pET16b、pET32a和p GEX-4T-1表达质粒构建pET16b-ybf E、pET32a-ybf E和p GEX-4T-1-ybf E原核表达载体,分别转化大肠杆菌BL21,IPTG诱导表达Ybf E融合蛋白,对三种表达系统的表达水平进行对比,并对pET16b-ybf E和pGEX-4T-1-ybf E表达体系的裂菌上清中的可溶性Ybf E融合蛋白液分别使用镍柱和GST蛋白纯化柱纯化。[结果]构建了pET16b-ybf E、pET32a-ybf E和p GEX-4T-1-ybf E原核表达体系,并使用IPTG诱导表达Ybf E融合蛋白。ybf E在p GEX-4T-1载体内的表达水平最高,接下来依次为pET16b和pET32a。pET16b-ybf E和p GEX-4T-1-ybf E表达的可溶性Ybf E融合蛋白纯化后浓度分别为86μg/m L和724μg/m L。[结论]成功构建了ybf E基因的三种原核表达系统,筛选出最佳表达体系,可溶性Ybf E融合蛋白得到纯化。     

EB病毒融合蛋白Zta-P54在大肠杆菌中的表达、纯化及鉴定

为获得能用于检测试剂盒的高纯度具有活性的EB病毒融合蛋白,以pGEX5T-BZLF1-BMRF1质粒为模板进行PCR扩增得到BZLF1-BMRF1融合基因,将其插入pET32a中,构建表达质粒pET32a/BZLF1-BMRF1。将该质粒转入大肠杆菌培养,经IPTG诱导获得Zta-P54融合蛋白。用DEAE-Sepharose CL-6B和Ni-NTA亲和层析纯化,并通过SDS-PAGE和Western blot对Zta-P54融合蛋白进行鉴定。双酶切鉴定和测序结果显示成功构建pET32a/BZLF1-BMRF1质粒,SDS-PAGE显示该蛋白相对分子量约为60kD,与预期结果一致。纯化后获得纯度为96.5%的Zta-P54融合蛋白。Western blot检测该蛋白有良好的生物活性和反应特异性。因此,成功构建pET32a/BZLF1-BMRF1质粒,在大肠杆菌中可溶性表达。最终获得纯度高、生物活性好的融合蛋白。     

Curcuminoids as inhibitors of thioredoxin reductase: A receptor based pharmacophore study with distance mapping of the active site

Curcumin is the yellow pigment of turmeric that interacts irreversibly forming an adduct with thioredoxin reductase (TrxR), an enzyme responsible for redox control of cell and defence against oxidative stress. Docking at both the active sites of TrxR was performed to compare the potency of three naturally occurring curcuminoids, namely curcumin, demethoxy curcumin and bis-demethoxy curcumin. Results show that active sites of TrxR occur at the junction of E and F chains. Volume and area of both cavities is predicted. It has been concluded by distance mapping of the most active conformations that Se atom of catalytic residue SeCYS498, is at a distance of 3.56 from C13 of demethoxy curcumin at the E chain active site, whereas C13 carbon atom forms adduct with Se atom of SeCys 498. We report that at least one methoxy group in curcuminoids is necessary for interation with catalytic residues of thioredoxin. Pharmacophore of both active sites of the TrxR receptor for curcumin and demethoxy curcumin molecules has been drawn and proposed for design and synthesis of most probable potent antiproliferative synthetic drugs.     

A Unique Protease Cleavage Site Predicted in the Spike Protein of the Novel Pneumonia Coronavirus (2019-nCoV) Potentially Related to Viral Transmissibility

正Dear Editor,In December 2019, a novel human coronavirus caused an epidemic of severe pneumonia(Coronavirus Disease 2019,COVID-19) in Wuhan, Hubei, China(Wu et al. 2020; Zhu et al. 2020). So far, this virus has spread to all areas of China and even to other countries. The epidemic has caused 67,102 confirmed infections with 1526 fatal cases     

Comparative morphology of the carpel in the Liliaceae: Hewardieae, Petrosavieae, and Tricyrteae

The young pistils in the melanthioid tribes, Hewardieae, Petrosavieae and Tricyrteae, are uniformly tricarpellate and syncarpous. They lack raphide idioblasts. All are multiovulate, with bitegmic ovules. The Petrosavieae are marked by the presence of septal glands and incomplete syncarpy. Tepals and stamens adhere to the ovary in the Hewardieae and the Petrosavieae but not in the Tricyrteae. Two vascular bundles occur in the stamens of the Hewartlieae and Tricyrtis latifolia. Ventral bundles in the upper part of the ovary of the Hewardieae are continuous with compound septal bundles and placental bundles in the lower part. Putative ventral bundles occur in the alternate position in the Tricyrteae and putative placental bundles in the opposite. position in the Petrosavieae. The dichtomously branched stigma in each carpel of the Tricyrteae is supplied by a bifurcated dorsal bundle.     

鸡传染性法氏囊病病毒研究进展

传染性法氏囊病(Infection bursal disease, IBD)是由鸡传染性法氏囊病毒(Infectious bursal disease virus, IBDV)引起的鸡和火鸡的一种高度接触性传染病,给世界各国的禽养殖业带来了巨大损失.自IBDV发现至今新的变异株不断出现,分子结构的改变导致病毒致病力的改变及宿主对疫苗应答的改变,使得传统的疫苗已不能控制其流行,因此各国学者对其基因组结构和功能进行了广泛深入的研究,并积极研制新型有效的疫苗以达到防治的目的.     

Development of a Novel Reverse Transcription Loop-Mediated Isothermal Amplification Method for Rapid Detection of SARS-CoV-2

In conclusion, the novel visual RT-LAMP assay is a simple, rapid, and sensitive approach for detection of SARS-CoV-2, and it is ready for application in primary care and community hospitals or health care centers, and even patients' own houses in response to the current SARS-CoV-2 epidemic because the assay does not require sophisticated equipment and skilled personnel. Furthermore, it is also ready to be used in fields for screening samples from wild animals and environments to facilitate the identification of potential intermediate hosts that mediate the cross-species transmission of SARS-CoV-2 from bats to humans.     

Perinatal Vertical Transmission of Chikungunya Virus in Ruili,a Town on the Border between China and Myanmar

正Dear Editor,Chikungunya virus (CHIKV), an arbovirus in the family of Togaviridae, genus Alphavirus, is transmitted by the A.aegyptii or A. albopictus mosquito, and causes disease in humans characterized by fever, rash, and arthralgia (Silva and Dermody 2017; Suhrbier 2019). It was first reported in 1953 in Tanzania, and caused only a few outbreaks and sporadic cases in Africa and Asia in last century. However, in the epidemic in 2004, CHIKV acquired mutations that conferred enhanced transmission by the A. albopictus mosquito(Schuffenecker et al. 2006). Since then, it has successively caused outbreaks in Africa, the Indian Ocean, South East Asia, the South America, and Europe (Zeller et al. 2016).     

Enterovirus 71 3C proteolytically processes the histone H3 N-terminal tail during infection

Highlights
1. The N-terminal tail of histone H3 is specifically cleaved during EV71 infection.
2. Viral protease 3C is identified as a protease responsible for proteolytically processing the N-terminal H3 tail.
3. Our finding reveals a new epigenetic regulatory mechanism for Enterovirus 71 in virus-host interactions.     

Detection of EBV and HHV6 in the Brain Tissue of Patients with Rasmussen’s Encephalitis

Rasmussen’s encephalitis (RE) is a rare pediatric neurological disorder, and the exact etiology is not clear. Viral infection may be involved in the pathogenesis of RE, but conflicting results have reported. In this study, we evaluated the expression of both Epstein-Barr virus (EBV) and human herpes virus (HHV) 6 antigens in brain sections from 30 patients with RE and 16 control individuals by immunohistochemistry. In the RE group, EBV and HHV6 antigens were detected in 56.7% (17/30) and 50% (15/30) of individuals, respectively. In contrast, no detectable EBV and HHV6 antigen expression was found in brain tissues of the control group. The co-expression of EBV and HHV6 was detected in 20.0% (6/30) of individuals. In particular, a 4-year-old boy had a typical clinical course, including a medical history of viral encephalitis, intractable epilepsy, and hemispheric atrophy. The co-expression of EBV and HHV6 was detected in neurons and astrocytes in the brain tissue, accompanied by a high frequency of CD8⁺ T cells. Our results suggest that EBV and HHV6 infection and the activation of CD8⁺ T cells are involved in the pathogenesis of RE.