信号肽疏水性的提高促进青霉素G酰化酶分泌

设计和合成了一段具有连续１０仆亮氨酸强疏水核心的信号肽（ａｒｔｉｆｉｃｉａｌ ｓｉｇｎａｌ ｐｅｐｔｉｄｅ,ＡＳＰ）,由ＥｃｏＲＩ－ＫｐｎＩ位点融合到青霉素Ｇ酰化酶（ｐｅｎｉｃｉｌｌｉｎ Ｇａｃｙｌａｓｅ,ＰＡＣ）信号肽（ｗｉｌｄ ｔｙｐｅｓｉｇｎａｌ ｐｅｐｔｉｄｅ,ＷＴＳＰ）的－４Ｐｒｏ位点,分别构建了ＰＡＣ表达质粒：ｐＫＫｐａｃ△ＳＰ,ｐＫＫｐａｃＷＴＳＰ,ｐＫＫｐａｃＡＳＰ,ｐＥＴｐａｃ     

异源nifA^C对根癌土壤杆菌nif基因表达的调节作用

用三亲水交配方法分别将载有褐球固氮菌（Ａｚｏｔｏｂａｃｔｅｒｃｈｒｏｏｃｏｃｃｕｍ）呈组成型表达的ｎｉｆＡＣ的质粒ｐＣＫ５和肺炎克氏杆菌（Ｋｌｅｂｓｉｅｌｌａｐｎｅｕｍｏｎｉａｅ）含有ｎｉｆＡ＾Ｃ和ｎｉｆＡ－ｎｔｒＣ基因的质粒ｐＣＫ３,ｐＳＺ３６和ｐＳＺ２３－ＣＡ导入根癌土壤杆菌（Ａｇｒｏｂａｃｔｅｒｉｕｍｔｕｎｅｆａｃｉｅｎｓ）Ｃ５８／ｐＧＶ３８５０所得转移接合子的生长速率和野生型相似。在１０ｍ     

胸苷激酶基因治疗胃癌的体外实验

将单纯疱疹病毒胸苷激酶基因（ＨＳＶ－ｔｋ）导入恶性肿瘤细胞,随后可应用药物丙氧鸟苷（ｇａｎｃｉｃｌｏｖｉｒ, ＧＣＶ）选择性杀死肿瘤细胞．构建了含胸苷激酶与潮霉素磷酸转移酶（ｈｐｈ）融和基因（ＨｙｔＫ）的真核表达载体ＬＸｐｓｐ－ＨｙｔＫ．以脂质体（ｌｉｐｏｆｅｃｔｉｎ）为介导,将这种质粒与仅含潮霉素Ｂ基因的质粒ＬＸＳＨ 分别转染胃癌细胞系ＢＧＣ－８２３,用６０ Ｕ／ｍ ｌ潮霉素Ｂ进行筛选,得到了可稳定传代的阳性克隆,分别命名为ＢＧＣ－ＨｙｔＫ 和ＢＧＣ－Ｈｙ．三种细胞的生长曲线无明显差别．用不同浓度的ＧＣＶ 分别作用于ＢＧＣ－ＨｙｔＫ, ＢＧＣ－Ｈｙ 及ＢＧＣ－８２３,０．０２～２００ μｇ／ｍ ｌ 的ＧＣＶ 对ＢＧＣ－ＨｙｔＫ 细胞有明显的杀伤作用（ＩＣ５０＝ ０．０２ μｇ／ｍ ｌ）,而对另外两种细胞几乎无毒性作用（ＩＣ５０＞ ２００μｇ／ｍ ｌ）．２０ μｇ／ｍ ｌＧＣＶ 作用９６ ｈ 后,仅存在２０％ 的ＢＧＣ－ＨｙｔＫ 就可使周围的大部分ＨＳＶ－ｔｋ－ 的肿瘤细胞死亡,说明存在较显著的“旁观者效应”     

B.thuringiensis穿梭载体的构建及cry1C基因的表达

以ＵＣ１９为母体,克隆了Ｂｔ ｋｅｎ－Ａｇ（Ｂ。．ｔｈｒｕｉｎｇｉｅｎｓｉｓ ｓｕｂｓｐ．ｋｅｎｙａｅ Ａｇ）的复制起始区（～１．６ｋｂ）、ｐＵＣ４Ｋ的ａｐｈ１基因,构建成穿梭载体ｐＨＶ－１,ｐＨＶ－１在Ｅ．ｃｏｌｉ中经１００个世代,质粒保持率在８０％以Ｂｔｉ ４Ｑ８（Ｂ．ｔｈｕｒｉｎｇｉｅｎｓｉｓ ｓｕｂｓｐ．ｉｓｒａｅｌｅｎｓｉｓ ４Ｑ８）中经４０个世代,质粒保持率在８０％以上,将Ｂ．ｌｉｃｈ     

水稻齿叶矮缩病毒Segment 9 dsRNA的序列分析及其在大肠杆菌DE3中的表达

通过有机试剂抽提,ＣＦ－１１纤维素柱层析,从感染水稻齿叶矮缩病毒菲律宾分离株（ＲｉｃｅＲａｇｇｅｄＳｔｕｎｔＶｉｒｕｓ,Ｐｈｉｌｉｐｐｉｎｅｉｓｏｌａｔｅ,简称ＲＲＳＶ－Ｐ）的水稻植株中获取该病毒的全基因组,即获得从Ｓｅｇｍｅｎｔ１到Ｓｅｇｍｅｎｔ１０（Ｓ１－Ｓ１０）的１０条双链ＲＮＡ（ｄｓＲＮＡ）,然后设计合适的引物,用ＲＴ－ＰＣＲ方法得到Ｓ９的ｃＤＮＡ并将其克隆到ｐＵＣ１１９质粒上扩增,以双链测序法测定该ｃＤＮＡ的全序列。同时又将此ｃＤＮＡ克隆到大肠杆菌表达质粒ｐＧＥＸ－３Ｘ上,在大肠杆菌菌株ＤＥ３中用ＩＰＴＧ诱导表达,经超声波破菌、离心、Ｇｌｕｔａｔｈｉｏｎｅ－ｓｅｐｈａｒｏｓｅ４Ｂ亲和层析,得到纯化的分子量为６４ｋＤ的融合蛋白。     

Bcl——2基因表达对TNF及OA诱发的细胞编程死亡的不同效应

用ＴＮＦ和ＯＡ（Ｏｋａｄａｉｃａｃｉｄ）诱发人神经母细胞瘤ＳＫ细胞死亡,并证明细胞死亡为编程死亡（ＰｒｏｇｒｍｍｅｄＣｅｌｌＤｅａｔｈ,简称ＰＣＤ）。将编码Ｂｃｌ－２全长蛋白的ｃＤＮＡ植入ＰＪＸ４１ｎｅｏ载体中,使其表达由ＨＣＭＶ病毒起动子控制。形成的顺义（ｐＢｃｌ－２－Ｓ）及反义（ｐＢｃｌ－２－ＡＳ）表达质粒经转染导入ＳＫ细胞中获得稳定转染子。Ｗｅｓｔｅｒｎ印迹表明顺义转染子表达大量的２６ｋｄＢｃｌ－２蛋白,而反义转染子则不表达。增强表达的Ｂｃｌ－２蛋白能抑制由ＴＮＦ引发的ＰＣＤ,但不影响ＯＡ引发的ＰＣＤ,从而证明了Ｂｃｌ－２基因产物抗细胞死亡效应的特异性。     

日本血吸虫26kD抗原基因在BCG中的表达

研究了外源基因日本血吸虫２６ｋＤ抗原（Ｓｊ２６ＧＳＴ）在卡介苗（ｂａｃｉｌｕｓＣａｌｍｅｔｅ－Ｇｕｅｒｉｎ,ＢＣＧ）、耻垢分枝杆菌（Ｍ．ｓｍｅｇｍａｔｉｓ）和大肠杆菌（Ｅ．ｃｏｌｉ）中的表达．运用重组ＤＮＡ和聚合酶链反应（ＰＣＲ）等分子生物学技术,以表达Ｓｊ２６ＧＳＴ的Ｅ．ｃｏｌｉｐＧＥＸ衍生质粒为模板,经ＰＣＲ得到编码Ｓｊ２６ＧＳＴ的全长ｃＤＮＡ片段．将其按正确的阅读框顺序,克隆到人结核杆菌热休克蛋白（ｈｅａｔｓｈｏｃｋｐｒｏｔｅｉｎ,ＨＳＰ）７０的启动子下游,再将ＨＳＰ７０启动子和Ｓｊ２６ＧＳＴ基因一起亚克隆到Ｅ．ｃｏｌｉ－分枝杆菌穿梭质粒ｐＢＣＧ－２０００中,得到Ｅ．ｃｏｌｉ－分枝杆菌穿梭表达质粒ｐＢＣＧ－Ｓｊ２６．ｐＢＣＧ－Ｓｊ２６电转化入ＢＣＧ和Ｍ．ｓｍｅｇｍａｔｉｓｍｃ２１５５中表达Ｓｊ２６ＧＳＴ抗原,所表达的天然重组Ｓｊ２６ＧＳＴ（ｒＳｊ２６ＧＳＴ）为可溶性蛋白,在ＳＤＳ－ＰＡＧＥ上分子量为２６ｋＤ处可见明显的表达蛋白带．其表达量分别占ＢＣＧ和Ｍ．ｓｍｅｇｍａｔｉｓ菌体总蛋白的１５％和１０％．可见,Ｓｊ２６ＧＳＴ基因能在ＢＣＧ中高效表达．     

Ti质粒介导的磷酸烯醇式丙酮酸羧化酶cDNA转化烟草植株

将玉米Ｃ４－磷酸烯醇式丙酮酸羧化酶（ＰＥＰ羧化酶）ｃＤＮＡ亚克隆至穿梭质粒ｐＢｉｎ１９,通过在杆菌Ｔｉ质粒（ＬＢＡ４４０４）介导的时圆片共培养法将其转入Ｃ３植物烟草中。在获得的抗性转化植株中,８０％具有较强的ＮＰＴⅡ报道基因表达。Ｓｏｕｔｈｅｒｎ杂交表明Ｃ４－ＰＥＰ羧化酶ｃＤＮＡ已被整合到了烟草核基因组中。     

抗芜菁花叶病毒转基因甘蓝型油菜的研究

以子叶柄为材料,建立了甘蓝型油菜（ＢｒａｓｓｉｃａｎａｐｕｓＬ．）双低品种的再生体系。通过子叶柄与农杆菌（ＡｇｒｏｂａｃｔｅｒｉｕｍｔｕｍｅｆａｃｉｅｎｓＬＢＡ４４０４）共培养,将表达载体ｐＢＴｕ中芜菁花叶病毒外壳蛋白（ＴｕＭＶ－ＣＰ）基因以整合方式导入甘蓝型油菜,然后用卡那霉素进行筛选,获得了油菜再生植株。经ＰＣＲ特异性扩增、点杂交和Ｓｏｕｔｈｅｒｎ印迹分析,证明再生植株基因组ＤＮＡ中整合了ＴｕＭＶ－ＣＰ基因。攻毒实验表明,有ＴｕＭＶ－ＣＰ基因插入的工程油菜对ＴｕＭＶ均有不同程度的抗性。     

鸡肌球蛋白轻链激酶表达载体的构建

鸡肌球蛋白轻链激酶（ＭＬＣＫ）在调节平滑肌细胞收缩中具有重要作用。用ＰＣＲ产生构建所需的限制性内切酶Ｓａｌ Ｉ位点,将鸡ＭＬＣＫ ｃＤＮＡ插入质粒ｐＢＫｒｓｖ中,构建成ｐＢＫｒｓｖ－ＭＬＣＫ,并通过酶切图谱、ＳａｌＩ位点序列分析得到证实。重组质粒在大肠杆菌中获得表达。     

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.