抗镰刀菌单链抗体在大肠杆菌中可溶性表达条件的研究

通过优化诱导表达条件,实现抗镰刀菌单链抗体在大肠杆菌周质中高效可溶性表达。将抗镰刀菌单链抗体FvSG7转化到大肠杆菌XL1-Blue中,确定诱导表达培养基,在不同的诱导温度、诱导剂IPTG的浓度和诱导时间条件下培养重组大肠杆菌,通过Western杂交和ELISA检测分析单链抗体的可溶性表达情况以及抗体的活性。重组大肠杆菌培养至OD600nm为0.5时加入终浓度为0.1 mmol/L IPTG,25℃诱导表达2 h可获得最大量的可溶性单链抗体。通过对诱导温度、IPTG浓度和诱导时间等表达条件的优化,可以显著提高Fv SG7抗体在大肠杆菌XL1-Blue周质中的表达量。     

A型产气荚膜梭菌α毒素基因表达及其免疫保护作用的初步研究

利用PCR技术,从A型产气荚膜梭菌标准株染色体DNA中扩增出α毒素基因,构建了含α毒素基因的重组菌株BL21(DE3)(pXETA02)。经酶切鉴定和序列测定证实,构建的表达质粒pXETA02含有α毒素基因序列。经SDS-PAGE、Western blot分析和ELISA检测,重组菌株表达的α毒素蛋白能够被α毒素单抗识别。表达优化结果表明,以IPTG为诱导剂诱导α毒素表达的优化条件是:培养基pH 7.5,培养温度37℃,IPTG浓度0.8mmol/L,菌体生长密度OD600达到0.8时加入IPTG,诱导时间5h,此时α毒素蛋白表达量为34.83%。以乳糖为诱导剂诱导α毒素表达的优化条件是:培养基pH7.5、培养温度37℃,乳糖浓度0.1g/L,菌体生长密度OD600达到0.8时加入乳糖,诱导时间5h,α毒素蛋白表达量为23.82%。动物实验结果表明,用重组菌株α毒素蛋白免疫的小鼠可以抵抗1MLD的A型产气荚膜梭菌标准株C57-1毒素攻击。     

肝靶向肽-抗肿瘤肽CSP-MDA-7/IL-24融合基因原核表达条件优化

目的:优化肝靶向肽-抗肿瘤肽(CSP-MDA-7/IL-24)在大肠杆菌中诱导表达的相关条件。方法:通过考察CSP-MDA-7/IL-24重组菌生长曲线,选择合适的诱导时机,通过SDS-PAGE检测CSP-MDA-7/IL-24蛋白的表达量,单因素分析诱导时间、培养基p H值、诱导温度和诱导剂IPTG浓度,在此基础上各选取5个水平进行正交实验,摸索最佳诱导表达条件。结果:培养基p H7.0、IPTG浓度0.12 mmol/L、培养温度42℃、诱导表达4 h为重组蛋白的最佳表达条件。结论:为进一步制备重组蛋白CSP-MDA-7/IL-24及评价其生物活性奠定了基础。     

乳糖诱导重组多价人精子表位肽在大肠杆菌中的表达

旨在研究用乳糖替代IPTG作为诱导剂进行重组多价人精子抗原表位肽的表达及诱导表达的优化条件.在摇瓶发酵条件下,通过改变培养基组成、诱导时机、诱导温度、诱导剂浓度、诱导时间和诱导方式等条件,利用SDS-PAGE电泳和AlphaEase凝胶电泳图像分析系统,研究以上条件改变对GST-重组多价人精子抗原表位肽融合蛋白表达量的影响,并与IPTG诱导结果相比较.结果显示,在摇瓶试验中,最优表达条件为选用TB培养基,在菌体对数生长的中期进行诱导,诱导温度37℃、乳糖诱导终浓度3 mmol/L、诱导时间6 h.GST-重组多价人精子抗原表位肽融合蛋白的表达量占菌体总蛋白的30.5％,且主要以可溶形式表达,与IPTG的诱导结果相同.分批流加乳糖和一次性加入乳糖诱导,效果一样.乳糖可以替代IPTG作为诱导剂诱导GST-重组多价人精子抗原表位肽融合蛋白的表达,优化条件下可获得与IPTG相同的诱导表达效果.     

大肠杆菌肠毒素ST1-LTB融合基因表达条件的优化

目的:对已构建的含有大肠杆菌肠毒素ST1-LTB融合基因的重组菌株进行鉴定和表达条件优化。方法:采用限制性核酸内切酶酶切鉴定含ST1-LTB融合基因的重组质粒,同时用SDS-PAGE检测不同条件下ST1-LTB融合基因的表达情况。结果:酶切鉴定证实重组质粒pXSL1含有ST1-LTB融合基因且阅读框架正确,以IPTG为诱导剂诱导ST1-LTB融合基因表达的优化条件是:培养基pH7.5,培养温度37℃,IPTG浓度0.8mmol/L,菌体生长密度OD600达到0.8时加入IPTG,诱导时间5h,此时ST1-LTB融合蛋白表达量达35.2%。结论:实现ST1-LTB融合基因的高效表达,为大肠杆菌肠毒素双价基因工程菌苗的生产工艺研究提供了可靠的实验数据。     

HCMV pp65截短蛋白原核表达条件优化

为了提高HCMVpp65蛋白片段在大肠杆菌中表达量,研究了不同发酵条件对其表达的影响,包括培养基、接种量、温度、摇床转速、pH、诱导时间以及诱导剂IPTG使用浓度等。结果表明,以LB培养基为发酵液,按5%接种量,37℃培养3h后IPTG诱导5h,重组菌菌体生物量为0.6g/L,目的蛋白表达量达18mg/L。用3.7L发酵罐进行放大培养,菌体生物量达0.85g/L,最高目的蛋白表达量达到25mg/L。     

重组人抗凝血酶发酵条件的优化

目的:通过实验室发酵条件优化工作,实现在巴斯德毕赤酵母中高效表达重组人抗凝血酶。方法:在摇瓶培养条件下,应用正交实验方法考查人抗凝血酶重组毕赤酵母菌pPIC9K-AT-02的培养温度、培养基pH值、接种比例、甲醇补加间隔时间及甲醇补加浓度等5种因素对重组人抗凝血酶活性的影响,确定最优发酵条件。结果与结论:筛选出的最终发酵条件为培养温度30℃、培养基pH6.0、接种比例20%、甲醇补加间隔时间24 h、甲醇补加浓度2%,重组人抗凝血酶在巴斯德毕赤酵母中表达活性为4098 U/L,比原始活性提高了150%。     

GABAA受体蛋白片段在大肠杆菌细胞表达条件研究

为了提高GABAA受体α1蛋白片段在大肠杆菌中表达量,研究了重组菌的发酵条件,包括培养基,接种量,温度,摇床转速,pH,诱导培养时间和诱导剂IPTG使用浓度等对GABAA受体蛋白片段表达的影响。结果表明重组菌以LB培养基为发酵基质,按3%接种量,37℃培养细胞3.5h后IPTG32℃诱导5h,菌体生物量为3.25g/L,目标蛋白表达量达95mg/L。用16L发酵罐进行放大培养,菌体生物量达4.95g/L,发酵周期5.5h。最高目标蛋白表达量达到136mg/L。     

运用均匀设计法优化rPA(K)在原核系统中的表达条件

运用均匀设计法对影响rPA(K)在大肠杆菌中表达的因素进行了优化设计,摸索出了对表达产物进行诱导表达的最佳优化表达条件。结果如下：溶解氧为90%,IPTG诱导时间为5.3 h、IPTG终浓度为0.1 mmol/L、培养基为HD、pH值为6.0、诱导温度为25?C。目的蛋白平均密度从0.16提高到0.48,是优化前的3倍。     

GABAA受体蛋白片段在大肠杆菌细胞表达条件研究*

为了提高GABAA受体a1蛋白片段在大肠杆菌中表达量,研究了重组菌的发酵条件,包括培养基、接种量、温度、摇床转速、pH、诱导培养时间和诱导剂IPTG使用浓度等对GABAA受体蛋白片段表达的影响。结果表明重组菌以LB培养基为发酵基质,按3％接种量,37℃培养细胞3.5h后IPTG 32℃诱导5h,菌体生物量为3.25g/L,目标蛋白表达量达95mg/L。用16L发酵罐进行放大培养,菌体生物量达4.95g/L,发酵周期5.5h,最高目标蛋白表达量达到136mg/L.     

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.