含PR启动子的原核高效表达载体的构建及应用

组建了一个仅含PR启动子的原核高效表达载体pRC,它同时含有cⅠ调控基因、多酶切位点和两个强的转录终止序列.现已成功地用于表达重组人肿瘤坏死因子-α(hTNF-α)、重组人白细胞介素-3(hIL-3)和抗溶菌酶(HEL)抗体Fd基因,表达量均占菌体总蛋白的36%以上.同时还研究了不同的宿主菌和原核增强子序列等因素对PR启动子载体表达的影响.此外,还比较了分别以PR、PL或PRPL作为启动子时表达hTNF-α的情况,结果表明,单用PR或PL启动子可获得与使用PRPL串联启动子一样的高效表达.     

小球藻病毒基因调控序列在大肠杆菌和真核藻中的调控活性研究

以小球藻病毒腺嘌呤甲基转移酶基因(amt)和主要外壳蛋白VP54基因的5′上游调控序列构建大肠杆菌和真核藻转化载体。以P_RP_L及CaMV35S启动子为阳性对照,研究了小球藻病毒来源的两种调控序列在E.coli和真核藻细胞中的启动活性。发现P_AMT在4种E.coli菌株中都具有极强的调控活性,启动Luc基因表达而产生的酶活性高于P_RP_L 50～400倍;P_VP54在DH5α中也具有较强的启动活性。同时PAMT在两种小球藻中启动GUS基因瞬时表达的能力也明显高于CaMV35S启动子,表明它们有可能在真核藻类遗传转化中具有很好的应用前景。     

中国人γ－干扰素cDNA在大肠杆菌中的高效表达

应用RT-PCR技术从中国人淋巴细胞mRNA反转录产物中克隆了IFN-γcDNA,序列分析证实了分子进化规律对IFN-γcDNA序列存在多态性的推论.在此基础上应用DNA重组技术,将去信号肽中国人IFN-γcDNA克隆到原核表达质粒pBV220 P_RP_L启动子下游,转化大肠杆菌DH5α,通过温度诱导表达,成功地在大肠杆菌中稳定、高效地表达了中国人IFN-γcDNA,其表达水平占全菌可溶性总蛋白的44.4%,初步复性后生物学活性测定结果表明γ-IFN表达量为0.45×10⁷～2.34×10⁷单位/L.     

腺苷酸激酶基因在大肠杆菌中的可溶性高表达

报道了鸡肌腺苷酸激酶基因的克隆和在温控启动子P_RP_L调控下在大肠杆菌中的可溶性高效表达.SDS-PAGE分析表明,鸡肌腺苷酸激酶的含量可占大肠杆菌细胞总蛋白含量的38％.利用Johnson等的干冰/乙醇-冰水浴反复冻融法,可将此重组蛋白进行富集,纯度可达85％以上.鸡肌腺苷酸激酶可与抗兔肌腺苷酸激酶单克隆抗体产生强的交叉反应.     

基因工程鱼生长激素的生产研究

以P_RP_L,Trp启动子在大肠杆菌中表达鲤鱼生长激素重组DNA,经过高密度发酵.包涵体的提取,恢复天然结构。每升发酵可碍2g鱼生长激素。在上海地区以浸渍方法处理对虾苗并在人工饲料中添加鱼生长激素,提高虾苗的存活率并增加产量,提高了饲料的转化率。     

抗CEA单链抗体与链亲和素融合基因的表达

克隆分泌CEA杂交瘤细胞重链可变区（V_H）和轻链可变区（V_L）,以Linker连接V_H及V_L构建抗CEA单链抗体.同时以Spacer连接单链抗体和链亲和素,构建成功单链抗体和链亲和素融合基因,克隆该融合基因至原核表达载体,pET21a(+),经IPTG诱导表达出该双特异性融合蛋白.活性鉴定表明该融合蛋白具有结合CEA及生物素的双特异性.该融合蛋白在生物领域中有较广阔的应用前景.     

乙型肝炎病毒pre-S1区中和抗体可变区的原核表达

鼠源单克隆抗体MA18/7是特异识别乙型肝炎病毒 (HBV)pre S1抗原的中和抗体。为表达MA18/7的小分子抗体 ,将MA18/7的重链可变区基因(V_H)和轻链可变区(V_L)基因分别克隆到原核表达载体pTO-T7进行原核表达。结果V_H和V_L均以包涵体的形式高效表达 ,包涵体经过变性、复性及金属离子亲和纯化后获得纯度>90%的重组抗体片段。生物传感器、竞争ELISA和间接ELISA测定均显示 ,V_H 或V_L 均不能结合相应抗原 ,但二者体外混合后可迅速非共价结合形成具有良好活性的可变区抗体Fv,表明MA18/7的抗原结合活性区由重链可变区和轻链可变区共同组成.     

抗A型产气荚膜梭菌α毒素单链抗体基因的克隆和表达

应用RT PCR技术 ,从分泌具有中和活性的抗A型产气荚膜梭菌α毒素单克隆抗体的杂交瘤细胞中 ,扩增出抗体V_H 和V_L 基因 ,连接成ScFv基因 ,并将其克隆至pGEM T载体中构建了重组质粒pXScFv 2E3。经序列分析证实 ,V_H 和V_L 基因及linker基因拼接正确 ,基因全长为 726bp ,编码 242个氨基酸。随后将其定向克隆于表达载体pHOG21,转化至大肠杆菌XL1 BLUE筛选出表达菌株XL1 BLUE(pHOG 2E3)。经ELISA和SDS PAGE分析表明 ,在20℃用IPTG诱导培养时 ,表达的ScFv蛋白占菌体总蛋白的 25 %。并且ScFv基因表达产物能够中和α毒素的磷酯酶C活性     

人工合成人溶菌酶基因在大肠杆菌中表达

利用DNA重组技术生产自然界不能或难以得到的多肽产品用于医药、农业、食品工业等领域,目前在生物领域已有了很大的进展。近年来,科学工作者经体外合成人溶菌酶基因,经克隆、转化,在真核、原核细胞中获得表达^[1-3]。溶菌酶能水解革兰氏阳性菌细胞壁上的β-1,4糖苷键,在内部渗透压的作用下,细胞胀裂开[4,5]。它对有些革兰氏阴性菌,如埃希氏大肠杆菌、伤寒沙门氏菌,也有同样作用。因此此酶在食品,特别是在医药上具有重要意义。在这篇文章中,我们报道人工合成人溶酶的克隆和在 温控启动子P_RP_L调控下在大肠杆菌中的表达。     

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.