无标签人源性脂联素球状结构的表达、纯化与活性检测

目的:利用基工程方法构建无标签人性脂联素球状结构(gAd)基的核表达载体,并对重组蛋白进行诱导表达、纯化及鉴定.方法:从正常人脂肪组织里提取总 RNA,反转录合成 cDNA,经 PCR 扩增、酶切后连入pET-22b(+)载体构建重组质粒 pET-22b(+)-gAd,转化大肠杆菌 BL21(DE3)感受态细胞,经低温、低浓度 IPTG 诱导使其可溶性表达,采用硫酸铵沉淀、凝胶过滤层析和离子交换层析三步分离纯化,得到不带任何标签的人性gAd;运用 SDS-PAGE、Western 印迹、HPLC 对重组蛋白进行鉴定,通过对 AMP 激活的蛋白激酶(AMPK)的磷酸化水平检测纯化蛋白的生物学活性.结果:构建了核表达载体 pET-22b(+)-gAd,实现了人性 gAd 在核细胞中的可溶性表达,纯化的蛋白经 SDS-PAGE 和 Western 印迹分析证实为 gAd,HPLC 分析蛋白纯度达到95%以上;通过对 AMPK磷酸化水平的检测,证明纯化的 gAd 具有高生物学活性.结论:重组表达和纯化了无标签、高生物学活性的人性脂联素球状结构,为其进一步的理论研究、生产开发奠定了基础.     

重组A型产气荚膜梭菌α毒素C端片段的分离纯化

押在构建出高表达、高活性的A型产气荚膜梭菌α毒素保护性抗原工程菌株pBV/cpa408的基础上,对表达产物进行了分离纯化研究。诱导表达菌液离心后,超声破碎沉淀细菌,上清用80%饱和硫酸铵沉淀,沉淀蛋白经透析,上凝胶过滤层析柱分离纯化,得到纯度达95%以上的表达目的蛋白,经SDS-PAGE测定,相对分子质量为15.5×103,序列与文献报道的相符。     

重组干扰素-τ在大肠杆菌中的表达与纯化

目的:探讨干扰素(IFN)-τ在大肠杆菌中的表达及纯化。方法:含有IFN-τ基因的pBV220表达质粒转化大肠杆菌BL21,于42℃温控诱导重组菌表达IFN-τ。经过包涵体溶解、DEAE离子交换层析、硫酸铵沉淀及梯度透析,使重组IFN-τ获得纯化和复性。结果:诱导后的表达产物经SDS-PAGE分析,有相对分子质量约21000的条带。纯化复性后目的蛋白纯度可达90%。结论:工程菌可稳定高效地表达IFN-τ。硫酸铵沉淀结合阴离子交换是一种简便高效的纯化方法,可获得较高纯度的IFN-τ。     

洋葱伯克霍尔德氏菌产邻苯二酚2,3-双加氧酶的研究

对洋葱伯克霍尔德氏菌L 68生长及产邻苯二酚2,3 双加氧酶(C23D)的条件进行了研究,其最适产酶pH7.2;最适生长温度30～35℃;最适培养时间48h;苯酚浓度0.09%最有利于菌体产酶.对菌株L 68产生的C23D酶进行了纯化,超声波破碎后的细胞提取液经硫酸铵分级沉淀、DEAESepharoseFastFlow层析、Hydroxyapatite层析、SephadexG 150层析后,收率为20%,酶比活力提高了230倍.SDS PAGE检测得到了分子量为(34±1)kDa的蛋白.     

重组A型产气荚膜梭菌α毒素保护性抗原的制备及初步免疫保护作用研究

诱导表达重组工程菌Pbv/cpa408后,将表达菌体超声破碎,上清经80%饱和硫酸铵一次沉淀,经透析,上凝胶过滤层析柱进行分离纯化,薄层凝胶扫描结果显示,纯化的蛋白纯度达95%以上;用纯化蛋白免疫昆明小鼠,以1.0MLD100腹腔进行攻击,被免疫小鼠获得了100%的保护。     

特异识别HIV-1序列的锌指蛋白的表达纯化及其在Biacore CM-5芯片上的固定

目的:为了更好地利用Biacore 3000研究锌指与核酸的相互作用,将特异性识别HIV-15′端一段保守序列的三锌指蛋白固定在CM-5芯片上。方法:将特异性识别HIV-15′端一段保守序列5′-CTGTGTTTG-3′的三锌指基因克隆到表达载体pET-22b( )中,转化大肠杆菌BL21(DE3)菌株,经IPTG诱导表达重组三锌指蛋白,超声碎菌进行SDS-PAGE分析;包涵体形式的表达产物用盐酸胍溶解后,经一步凝胶柱复性并纯化;随后摸索适宜固定的pH值并通过化学方法进行固定。结果:表达的重组蛋白主要以包涵体形式存在于超声沉淀中,纯化及柱复性后的蛋白纯度为98.8%,并在CM-5芯片上成功固定。结论:本研究为利用Biacore实时定量研究锌指蛋白与其识别DNA的相互作用进行了尝试。     

融合干扰素-BLA（IFN-BLA）的构建、表达、纯化及抗病毒活性测定

干扰素-BLA(IFN-BLA)由干扰素-beta-1b(IFN-beta-1b)和干扰素-alpha-2b（IFN-alpha-2b）通过连接肽-GGGS-融合而成。优化了其在大肠感菌BL21 CodonPlus (DE3)-RIL中的实验室表达条件,表达的目的蛋白占菌体总蛋白的35％以上并且主要以包涵体的形式存在。对包涵体的复性条件进行了摸索,建立了IFN-BLA的复性及纯化方法,纯化后的蛋白产量约为45 mg/L, 纯度在90％以上。抗病毒活性分析表明这一新的融合蛋白可能具有协同或加成活性。     

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。     

头孢菌素脱乙酰酶的重组表达及固定化

本文构建了利用trp启动子表达头孢菌素脱乙酰酶(CAH)的重组大肠杆菌DH5α-pCAH。重组菌在7L发酵罐(装液量2L)中发酵28 h,发酵液OD_(600)达到27,产酶313 kU/L发酵液,粗略估算重组蛋白占细胞总蛋白的70%。发酵生产的重组CAH粗酶液经过硫酸铵分级沉淀分离纯化和超滤除盐浓缩两步操作,纯化倍数为1.44,总酶活回收率56%,聚丙烯酰胺凝胶电泳检测纯化后蛋白没有明显杂蛋白条带出现。纯化后的CAH共价结合固定在环氧基载体LX-1000EP(c)上,通过对固定化条件的优化最终得到固定化酶比活443 U/g。该固定化酶重复催化50 mL 5%7-ACA底物100次后,酶活没有降低。     

B型肉毒毒素轻链的高效制备及活性鉴定

目的:在大肠杆菌中高效表达B型肉毒毒素轻链(BoNT/BLC)并纯化,研究其生物学活性。方法:根据报道的BoNT/B LC基因序列设计引物,从肉毒梭菌中扩增BoNT/BLC基因片段,将其克隆至原核表达载体pET-22b中,重组质粒转化大肠杆菌BL21(DE3)Rosetta感受态细胞,构建重组大肠杆菌pET-22b BoNT/BLC/BL21(DE3)Rosetta,在20℃条件下用IPTG诱导目的蛋白表达,表达产物经His Trap FF柱纯化,用SDS-PAGE对目的蛋白进行鉴定,并利用相应底物对纯化产物进行生物活性分析。结果与结论:构建了重组大肠杆菌pET-22b BoNT/BLC/BL21(DE3)Rosetta,BoNT/BLC表达量达到了细菌总蛋白的30%左右,通过一步亲和纯化目的蛋白后经SDS-PAGE检测其纯度在95%以上,制备的重组LC的酶活略高于B型肉毒毒素全毒素,可作为试剂用于BoNT/BLC抑制剂高通量体外检测方法的研究。     

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     

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.     

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.     

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.     

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).     

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.