高山被孢霉ATCC16266Δ~6-脂肪酸脱氢酶基因在酿酒酵母中的表达

Δ6 脂肪酸脱氢酶是形成γ 亚麻酸的关键酶。从含有高山被孢霉Δ6 脂肪酸脱氢酶基因的重组质粒pT MACL6中 ,酶切出 1 4kb的目的片段 ,亚克隆到大肠杆菌和酿酒酵母的穿梭表达载体 pYES2 .0 ,在大肠杆菌中筛选到含有目的基因的重组质粒 pYMAD6 ,用醋酸锂方法转化到酿酒酵母的缺陷型菌株INCSc1中 ,在SC Ura合成培养基中 ,选择得到酿酒酵母工程株YMAD6。在合适的培养基及培养条件下 ,加入外源底物亚油酸 ,经半乳糖诱导后 ,收集菌体。通过GC MS对酵母工程株进行脂肪酸色谱分析 ,结果表明 ,产生了 31 6 %的γ 亚麻酸。这是迄今为止 ,国内外Δ6 脂肪酸脱氢酶基因在酿酒酵母中表达量最高的报道。     

HIV-1 gp41的酵母表面展示及表达优化

以His标签检测蛋白的表达, 利用酿酒酵母表面展示系统, 成功地将HIV-1 gp41片段锚定在酵母表面, 并检测到gp41的活性。以pMD18T-gp41为模板, 通过PCR技术克隆了gp41基因, 将gp41基因通过双酶切连接到载体pICAS-His上,构建了gp41酵母表面展示载体, 并将其转化至酿酒酵母(Saccharomyces cerevisiae)MT8-1中。重组菌经培养, 利用免疫荧光染色方法进行染色, 显微镜观察发现重组酵母细胞表面有绿色荧光, 流式细胞仪结果进一步证实gp41正确折叠展示于酵母细胞表面。采用不同浓度的葡萄糖培养基进行表达优化。当葡萄糖浓度为1%时, 82.46%的酵母细胞表达了gp41抗原; 随着葡萄糖浓度升高, 蛋白表达受到抑制。     

HIV-1 gp41的酵母表面展示及表达优化

以His标签检测蛋白的表达, 利用酿酒酵母表面展示系统, 成功地将HIV-1 gp41片段锚定在酵母表面, 并检测到gp41的活性。以pMD18T-gp41为模板, 通过PCR技术克隆了gp41基因, 将gp41基因通过双酶切连接到载体pICAS-His上,构建了gp41酵母表面展示载体, 并将其转化至酿酒酵母(Saccharomyces cerevisiae)MT8-1中。重组菌经培养, 利用免疫荧光染色方法进行染色, 显微镜观察发现重组酵母细胞表面有绿色荧光, 流式细胞仪结果进一步证实gp41正确折叠展示于酵母细胞表面。采用不同浓度的葡萄糖培养基进行表达优化。当葡萄糖浓度为1%时, 82.46%的酵母细胞表达了gp41抗原; 随着葡萄糖浓度升高, 蛋白表达受到抑制。     

高山被孢霉ATCC16266Δ6-脂肪酸脱氢酶基因在酿酒酵母中的表达

Δ⁶-脂肪酸脱氢酶是形成γ-亚麻酸的关键酶。从含有高山被孢霉Δ⁶-脂肪酸脱氢酶基因的重组质粒pTMACL6中,酶切出14kb的目的片段,亚克隆到大肠杆菌和酿酒酵母的穿梭表达载体pYES2.0,在大肠杆菌中筛选到含有目的基因的重组质粒pYMAD6,用醋酸锂方法转化到酿酒酵母的缺陷型菌株INCSc1中,在SC-Ura合成培养基中,选择得到酿酒酵母工程株YMAD6。在合适的培养基及培养条件下,加入外源底物亚油酸,经半乳糖诱导后,收集菌体。通过GC-MS对酵母工程株进行脂肪酸色谱分析,结果表明,产生了31.6%的γ-亚麻酸。这是迄今为止,国内外Δ⁶-脂肪酸脱氢酶基因在酿酒酵母中表达量最高的报道。     

深黄被孢霉M_(6-22) Δ~6-脂肪酸脱氢酶基因在酿酒酵母中的表达

应用PCR技术 ,从含有深黄被孢霉Δ6 脂肪酸脱氢酶基因的重组质粒pTMICL6中 ,扩增出 1 38kb的目的片段 ,亚克隆到大肠杆菌和酿酒酵母的穿梭表达载体pYES2 .0 ,在大肠杆菌中筛选到含有目的基因的重组质粒pYMID6,用醋酸锂方法转化到酿酒酵母的缺陷型菌株INVSc1中 ,在SC Ura合成培养基中 ,选择到酵母工程株YMID6。在合适的培养基及培养条件下 ,加入外源底物亚油酸 ,经半乳糖诱导后 ,收集菌体。通过GC MS对酵母工程株所含的全部脂肪酸进行色谱分析 ,结果表明 ,γ 亚麻酸的含量占酵母总脂肪酸的 8 69%。     

海洋破囊壶菌Δ~4-脂肪酸脱饱和酶基因在酿酒酵母中的表达

以质粒pGEM-TFAD4为模板,扩增获得1.6kb的Δ4-脂肪酸脱饱和酶基因(FAD4)。将FAD4酶切后连接到HindⅢ/XbaⅠ处理过的pYES2.0载体,构建重组表达质粒pYFAD4。转化酿酒酵母缺陷型菌INVScl,通过SC-U选择性培养基筛选阳性克隆子。添加外源脂肪酸C22:5底物,半乳糖诱导表达。气相色谱分析表明阳性克隆子总脂肪酸中出现了二十二碳六烯酸C22:6(占酵母总脂肪含量的41.13%),Δ4-脂肪酸脱饱和酶基因在酿酒酵母中得到了表达。     

酿酒酵母乙醇脱氢酶2(ADH2)基因的克隆表达及活性验证

为了从酿酒酵母Saccharomyces cerevisiae中克隆出乙醇脱氢酶2(Alcoholdehy drogenase2,ADH2)基因并使之在大肠杆菌中高效表达。以酿酒酵母细胞中提取的总RNA为模板,通过反转录获得酿酒酵母乙醇脱氢酶2基因,连接到表达载体pTAT上,得到重组表达质粒pTAT-ADH2,将此重组质粒转化到大肠杆菌BL21中,重组工程菌株经IPTG诱导表达得到ADH2蛋白。将该蛋白纯化后,在体外进行活性检测和小鼠体内进行毒理试验,检测ADH2的酶活性。测序结果表明克隆的基因与GenBank中所报道的adh2基因序列有90%的同源性,经SDS-PAGE电泳分析,目的蛋白得到了有效表达,蛋白条带扫描分析表明,表达量占总蛋白的50%左右,纯化得到的蛋白在小鼠体内进行毒理试验,显示出一定的活性。酿酒酵母adh2基因的克隆正确,不仅在大肠杆菌中进行了高效表达而且表现出了较好的酶活性。     

卷枝毛霉Δ6-脂肪酸脱氢酶基因的克隆及在酿酒酵母中的高效表达

为获得产高γ 亚麻酸的酿酒酵母工程菌株,应用RT PCR技术,从卷枝毛霉中扩增出△6 脂肪酸脱氢酶 基因,亚克隆到大肠杆菌和酿酒酵母的穿梭表达载体pYES2.0,在大肠杆菌中筛选到含有目的基因的重组质粒 pYES412,用醋酸锂方法转化到酿酒酵母缺陷型菌株INVScI中,在SC ura合成培养基中筛选到转化酵母,在合适 的培养基及培养条件下,加入外源底物亚油酸,经半乳糖诱导后,收集菌体,通过气相色谱对转化酵母进行脂肪酸 色谱分析,结果表明:γ 亚麻酸占总脂肪的50.07%。迄今为止,这是国内外△6 脂肪酸脱氢酶基因在酿酒酵母表 达量最高的报道。     

深黄被孢霉M6-22

应用PCR技术,从含有深黄被孢霉△6-脂肪酸脱氢酶基因的重组质粒pTMICL6中,扩增出1.38kb的目的片段,亚克隆到大肠杆菌和酿酒酵母的穿梭表达载体pYES2.0,在大肠杆菌中筛选到含有目的基因的重组质粒pYMID6,用醋酸锂方法转化到酿酒酵母的缺陷型菌株INVSc1中,在SC-Ura合成培养基中,选择到酵母工程株YMID6.在合适的培养基及培养条件下,加入外源底物亚油酸,经半乳糖诱导后,收集菌体.通过GC-MS对酵母工程株所含的全部脂肪酸进行色谱分析,结果表明,γ-亚麻酸的含量占酵母总脂肪酸的8.69%.     

深黄被孢霉M6-22△6-脂肪酸脱氢酶基因在酿酒酵母中的表达

应用PCR技术,从含有深黄被孢霉△6-脂肪酸脱氢酶基因的重组质粒pTMICL6中,扩增出1.38kb的目的片段,亚克隆到大肠杆菌和酿酒酵母的穿梭表达载体pYES2.0,在大肠杆菌中筛选到含有目的基因的重组质粒pYMID6,用醋酸锂方法转化到酿酒酵母的缺陷型菌株INVSc1中,在SC-Ura合成培养基中,选择到酵母工程株YMID6.在合适的培养基及培养条件下,加入外源底物亚油酸,经半乳糖诱导后,收集菌体.通过GC-MS对酵母工程株所含的全部脂肪酸进行色谱分析,结果表明,γ-亚麻酸的含量占酵母总脂肪酸的8.69%.     

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.