lncRNA SNHG4 enhanced gastric cancer progression by modulating miR-409-3p/CREB1 axis

Objective:Gastric cancer(GC)is a globally common cancer characterized by high incidence and mortality worldwide.Advances in the molecular understanding of GC provide promising targets for GC diagnosis and therapy.Long non-coding RNAs(lncRNAs)and their downstream regulators are regarded to be implicated in the progression of multiple types of malignancies.Studies have shown that the lncRNA small nucleolar RNA host gene 4(SNHG4)serves as a tumor promoter in various malignancies,while its function in GC has yet to be characterized.Therefore,our study aimed to explore the role and underlying mechanism of SNHG4 in GC.Methods:We used qRT-PCR to analyze SNHG4 expression in GC tissues and cells.Kaplan-Meier analysis was used to assess the correlation between SNHG4 expression and the survival rate of GC patients.Cellular function experiments such as CCK-8,BrdU,colony formation,flow cytometry analysis,and transwell were performed to explore the effects of SNHG4 on GC cell proliferation,apoptosis,cell cycle,migration,and invasion.We also established xenograft mouse models to explore the effect of SNHG4 on GC tumor growth.Mechanically,dual luciferase reporter assay was used to verify the interaction between SNHG4 and miR-409-3p and between miR-409-3p and cAMP responsive element binding protein 1(CREB1).Results:The results indicated that SNHG4 was overexpressed in GC tissues and cell lines,and was linked with poor survival rate of GC patients.SNHG4 promoted GC cell proliferation,migration,and invasion while inhibiting cell apoptosis and cell cycle arrest in vitro.The in vivo experiment indicated that SNHG4 facilitated GC tumor growth.Furthermore,SNHG4 was demonstrated to bind to miR-409-3p.Moreover,CREB1 was directly targeted by miR-409-3p.Rescue assays demonstrated that miR-409-3p deficiency reversed the suppressive impact of SNHG4 knockdown on GC cell malignancy.Additionally,miR-409-3p was also revealed to inhibit GC cell proliferation,migration,and invasion by targeting CREB1.Conclusion:In conclusion,we verified that the SNHG4 promoted GC growth and metastasis by binding to miR-409-3p to upregulate CREB1,which may deepen the understanding of the underlying mechanism in GC development.

SMAD specific E3 ubiquitin protein ligase 1 accelerates diabetic macular edema progression by WNT inhibitory factor 1

BACKGROUND Diabetic macular edema(DME)is the most common cause of vision loss in people with diabetes.Tight junction disruption of the retinal pigment epithelium(RPE)cells has been reported to induce DME development.SMAD-specific E3 ubiquitin protein ligase(SMURF)1 was associated with the tight junctions of cells.However,the mechanism of SMURF1 in the DME process remains unclear.AIM To investigate the role of SMURF1 in RPE cell tight junction during DME.METHODS ARPE-19 cells treated with high glucose(HG)and desferrioxamine mesylate(DFX)for establishment of the DME cell model.DME mice models were constructed by streptozotocin induction.The trans-epithelial electrical resistance and permeability of RPE cells were analyzed.The expressions of tight junction-related and autophagy-related proteins were determined.The interaction between insulin like growth factor 2 mRNA binding protein 2(IGF2BP2)and SMURF1 mRNA was verified by RNA immunoprecipitation(RIP).SMURF1 N6-methyladenosine(m6A)level was detected by methylated RIP.RESULTS SMURF1 and vascular endothelial growth factor(VEGF)were upregulated in DME.SMURF1 knockdown reduced HG/DFX-induced autophagy,which protected RPE cell tight junctions and ameliorated retinal damage in DME mice.SMURF1 activated the Wnt/β-catenin-VEGF signaling pathway by promoting WNT inhibitory factor(WIF)1 ubiquitination and degradation.IGF2BP2 upregulated SMURF1 expression in an m6A modification-dependent manner.CONCLUSION M6A-modified SMURF1 promoted WIF1 ubiquitination and degradation,which activated autophagy to inhibit RPE cell tight junctions,ultimately promoting DME progression.

Construction of a Yeast Hybrid Library and Identification of Proteins Regulating CaABI3/VP1-1 Expression in Capsicum annuum var.conoides

Hot pepper(Capsicum annuum var.conoides)is a significant vegetable that is widely cultivated around the world.Currently,global climate change has caused frequent severe weather events,and waterlogging stress harms the pepper industry by affecting the planting period,growth conditions,and disease susceptibility.The gene CaABI3/VP1-1 could improve pepper waterlogging tolerance.In order to explore the upstream regulatory mechanism of CaABI3/VP1-1,a high-quality standardized yeast hybrid library was successfully constructed for yeast one-,two-,and threehybrid screening using pepper‘ZHC2’as the experimental material,with a library recombinant efficiency of up to 100%.The length of inserted fragments varied from 650 to 5000 bp,the library titer was 5.18×10^(6)colony-forming units(CFU)·mL-1,and the library capacity was 1.04×10^(7)CFU of cDNA inserts.The recombinant bait plasmid was used to successfully identify 78 different proteins through the yeast one-hybrid system,including one transcription factor within the ethylene-responsive factor family and the other within the growth-regulating factor family.The interaction happened between LOC124895848 and CaABI3/VP1-1 promoter by point-to-point yeast one-hybrid experiment.The expression level of the 12 selected protein-coding genes was then evaluated by quantitative real-time polymerase chain reaction.Results indicated the protein coding genes showed different responses to waterlogging stress and that the activity of the CaABI3/VP1-1 promoter could be inhibited or activated by up-regulating or down-regulating gene expression,respectively.The identification of these proteins interacting with the promoter provides a new perspective for understanding the gene regulatory network of hot pepper operating under waterlogging stress and provides theoretical support for further analysis of the complex regulatory relationship between transcription factors and promoters.

基于Wnt/β-连环蛋白通路探究金天格对肿瘤坏死因子-α诱导的小鼠MC3T3E1细胞生物学功能的影响实验研究

目的:探讨金天格通过调节Wnt/β-连环蛋白(Wnt/β-catenin)通路对肿瘤坏死因子-α(TNF-α)诱导的小鼠成骨细胞(MC3T3E1)细胞生物学功能的影响。方法:体外培养MC3T3E1细胞,分为对照组、TNF-α组(50 ng/ml TNF-α)、L-金天格组(50 ng/ml TNF-α+10^(-6) g/L金天格)、M-金天格组(50 ng/ml TNF-α+10-5 g/L金天格)、H-金天格组(50 ng/ml TNF-α+10^(-4) g/L金天格)、Dickkopf-1(DKK-1)组(50 ng/ml TNF-α+10 ng/ml Wnt/β-catenin通路抑制剂DKK-1)、H-金天格+LiCl组(50 ng/ml TNF-α+10^(-4) g/L金天格+20μmol/L Wnt/β-catenin通路激活剂LiCl)。用CCK-8试剂盒对细胞活性进行检测,用流式细胞仪对细胞凋亡情况进行检测,用酶联免疫吸附试验对细胞白细胞介素-1β(IL-1β)和IL-6水平进行检测,用Western blot对细胞凋亡相关蛋白及Wnt/β-catenin信号通路蛋白表达情况进行检测。结果:与对照组比较,TNF-α组细胞活性、B淋巴细胞瘤-2(Bcl-2)、细胞程序性死亡配体-1(PD-L1)蛋白表达降低,细胞凋亡率、IL-1β、IL-6水平以及B细胞淋巴瘤(Bax)、β-catenin、转录因子7样2(TCF7L2)、细胞周期蛋白D1(Cyclin D1)蛋白表达升高(均P<0.05)。与TNF-α组比较,L-金天格组、M-金天格组、H-金天格组、DKK-1组细胞活性及Bcl-2、PD-L1蛋白表达升高,细胞凋亡率、IL-1β、IL-6水平以及Bax、β-catenin、TCF7L2、Cyclin D1蛋白表达降低(均P<0.05)。与H-金天格组比较,H-金天格+LiCl组细胞活性及Bcl-2、PD-L1蛋白表达降低,细胞凋亡率、IL-1β、IL-6水平以及Bax、β-catenin、TCF7L2、Cyclin D1蛋白表达升高(均P<0.05)。结论:金天格可能通过抑制Wnt/β-catenin通路减轻TNF-α诱导的MC3T3E1细胞损伤。

SNHG3调控miR-186-5p/ZEB1促进肝癌细胞增殖、迁移、侵袭和上皮-间质转化的机制研究

目的探究SNHG3调控miR-186-5p/E盒结合锌指蛋白1(ZEB1)促进肝癌细胞增殖、迁移、侵袭和上皮-间质转化(EMT)的机制。方法在HepG2细胞中转染siRNA干扰片段敲除SNHG3、转染pcDNA3.1(+)/SNHG3使SNHG3过表达,通过实时荧光定量聚合酶链式反应(FQ-PCR)检测是否转染成功。克隆形成实验检测HepG2细胞增殖,细胞划痕实验检测HepG2细胞迁移率,Transwell小室实验检测HepG2细胞侵袭数量,双荧光素酶活性检测验证SNHG3与miR-186-5p、miR-186-5p与ZEB1的靶向关系,FQ-PCR和Western blot法检测SNHG3、ZEB1、E-cadherin、N-cadherin、MMP-2、vimentin、MMP-9、Snail的表达水平。结果肝癌组织中SNHG3表达量高于正常组织,SNHG3表达量低/中的肝癌患者的生存预后优于SNHG3表达量高的肝癌患者,差异有统计学意义(P<0.05)。克隆形成实验结果显示,与pcDNA3.1(+)组相比,pcDNA3.1(+)/SNHG3组HepG2细胞增殖数量显著增加(P<0.05)。细胞划痕实验结果显示,pcDNA3.1(+)/SNHG3组HepG2细胞迁移率显著高于pcDNA3.1(+)组(P<0.05)。Transwell小室实验结果显示,pcDNA3.1(+)/SNHG3组HepG2细胞侵袭数量显著高于pcDNA3.1(+)组(P<0.05)。双荧光素酶活性检测结果显示,miR-186-5p mimic和pGL6-SNHG3-WT共转染后,荧光素酶活性低于其他组,miR-186-5p mimic和pGL6-ZEB1-WT共转染后,荧光素酶活性低于其他组,差异有统计学意义(P<0.05)。FQ-PCR和Western blot检测结果显示,当SNHG3过表达时,E-cadherin mRNA和蛋白相对表达量显著下调,N-cadherin、MMP-2、vimentin、MMP-9和Snail mRNA和蛋白相对表达量以及ZEB1蛋白相对表达量显著上调;当SNHG3敲除后,E-cadherin mRNA和蛋白相对表达量显著上调,N-cadherin、MMP-2、vimentin、MMP-9和Snail mRNA和蛋白相对表达量以及ZEB1蛋白相对表达量显著下调,差异有统计学意义(P<0.05)。结论SNHG3调控miR-186-5p/ZEB1可促进肝癌细胞增殖、迁移、侵袭和EMT,表明SNHG3是肝癌潜在的治疗靶点。

Involvement of ERK1/2 and p38 MAPK in up-regulation of 14-3-3 protein induced by hydrogen peroxide preconditioning in PC12 cells

Objective To investigate the protective effects of hydrogen peroxide preconditioning （HPP） on the pheochromocytoma （PC12） cells treated with 1-methyl-4-phenylpyridinium （MPP^＋） and to explore the potential mechanisms. Methods The viability and apoptosis of PC 12 cells were determinded by 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide （MTT） assay and 4′,6′-diamidino-2-phenylindole （DAPI） staining, respectively. The expressions of 14-3-3 protein and phospholylated p38 mitogen-activated protein kinase （MAPK） were determined by Western blot. Enzyme-linked immunosorbent assay （ELISA） was used to measure the activity of extracellular signal-regulated protein kinase 1/2 （ERK1/2）. Results The cell viability decreased and the number of apoptotic cells increased dramatically in MPP^＋ group compared with that in Control group. HPP induced a significant increase in cell viability and a marked decrease in population of apoptotic cells of the MPP^＋- treated PC 12 cells, accompanied with up-regulation of 14-3-3 protein and increase of ERK 1/2 and p38 MAPK activities. The 14-3-3 protein expression was positively correlated with the phosphorylation of ERK1/2. Furthermore, inhibition of the ERK1/2 with PD98059 abolished the 14-3-3 protein up-regulation in PC 12 cells induced by HPP. Conclusion HPP protects PC 12 cells against MPP＋ toxicity by up-regulating 14-3-3 protein expression through the ERK1/2 and p38 MAPK signaling pathways.

Exosomes derived from microglia overexpressing miR-124-3p alleviate neuronal endoplasmic reticulum stress damage after repetitive mild traumatic brain injury

We previously reported that miR-124-3p is markedly upregulated in microglia-derived exosomes following repetitive mild traumatic brain injury.However,its impact on neuronal endoplasmic reticulum stress following repetitive mild traumatic brain injury remains unclear.In this study,we first used an HT22 scratch injury model to mimic traumatic brain injury,then co-cultured the HT22 cells with BV2 microglia expressing high levels of miR-124-3p.We found that exosomes containing high levels of miR-124-3p attenuated apoptosis and endoplasmic reticulum stress.Furthermore,luciferase reporter assay analysis confirmed that miR-124-3p bound specifically to the endoplasmic reticulum stress-related protein IRE1α,while an IRE1αfunctional salvage experiment confirmed that miR-124-3p targeted IRE1αand reduced its expression,thereby inhibiting endoplasmic reticulum stress in injured neurons.Finally,we delivered microglia-derived exosomes containing miR-124-3p intranasally to a mouse model of repetitive mild traumatic brain injury and found that endoplasmic reticulum stress and apoptosis levels in hippocampal neurons were significantly reduced.These findings suggest that,after repetitive mild traumatic brain injury,miR-124-3 can be transferred from microglia-derived exosomes to injured neurons,where it exerts a neuroprotective effect by inhibiting endoplasmic reticulum stress.Therefore,microglia-derived exosomes containing miR-124-3p may represent a novel therapeutic strategy for repetitive mild traumatic brain injury.

Home Monitoring of Estrone-3-Glucuronide (E1-3G) Levels in Two Different Ovarian Stimulation Protocols: A Pilot Study

Background: Studies have shown a strong correlation between the growth of E2 in serum and estrone-3-glucuronide (E1-3G) in urine during ovarian stimulation. Thus, we developed theoretical models for using urinary E1-3G in ovarian stimulation and focused on their experimental verification and analysis. Methods: A prospective, observational pilot study was conducted involving 54 patients who underwent 54 cycles of ovarian stimulation. The goal was to establish the growth rate of urinary E1-3G during the course of stimulation and to determine the daily upper and lower limits of growth rates at which stimulation is appropriate and safe. Controlled ovarian stimulation was performed using two different stimulation protocols—an antagonist protocol in 25 cases and a progestin-primed ovarian stimulation protocol (PPOS) in 29 cases, with fixed doses of gonadotropins. From the second day of stimulation, patients self-measured their daily urine E1-3G levels at home using a portable analyzer. In parallel, a standard ultrasound follow-up protocol accompanied by a determination of E2, LH, and P levels was applied to optimally control stimulation. Results: The average daily growth rates in both groups were about 50%. The daily increase in E1-3G for the antagonist protocol ranged from 14% to 79%, while they were 28% to 79% for the PPOS protocol. Conclusion: This is the first study to analyze the dynamics of E1-3G in two different protocols and to estimate the limits of its increase during the entire course of the stimulation. The results confirm our theoretical model for the viability of using urinary E1-3G for monitoring ovarian stimulation.

Rice leaf inclination2, a VIN3-1ike protein, regulates leaf angle through modulating cell division of the collar

As an important agronomic trait, inclination of leaves is crucial Ior crop architecture and grain yields. 10 understand the molecular mechanism controlling rice leaf angles, one rice leaf inclination2 （1c2, three alleles） mutant was identified and functionally characterized. Compared to wild-type plants, lc2 mutants have enlarged leaf angles due to increased cell division in the adaxial epidermis of lamina joint. The LC2 gene was isolated through positional cloning, and encodes a vernalization insensitive 3-like protein. Complementary expression of LC2 reversed the enlarged leaf angles of lc2 plants, confirming its role in controlling leaf inclination. LC2 is mainly expressed in the lamina joint during leaf development, and particularly, is induced by the phytohormones abscisic acid, gibberellic acid, auxin, and brassinosteroids. LC2 is localized in the nucleus and defects of LC2 result in altered expression of cell division and hormone-responsive genes, indicating an important role of LC2 in regulating leaf inclination and mediating hormone effects.

糖尿病足溃疡患者外周血Nrf2/HO-1/NLRP3炎性小体表达变化及临床意义

糖尿病足是糖尿病严重并发症之一,下肢血管病变形成微循环障碍,导致溃疡和坏疽[1]。糖尿病足溃疡(DFU)是糖尿病足最常见的表现形式,如若创面出现继发性感染,不及时控制病情,软组织感染将进一步侵袭骨质,造成骨髓炎,危及生命[2]。核苷酸结合寡聚化结构域样受体蛋白3(NLRP3)炎性小体是固有免疫的重要组分,在多种炎症性疾病的发生发展中发挥重要作用[3]。

鸡堆型艾美尔球虫3-1E抗原表位的生物信息学预测

应用生物信息学分析和预测鸡堆型艾美尔球虫3-1E蛋白二级结构和抗原表位,为确定和筛选优势表位,研制安全、高效表位疫苗奠定基础。以克隆获得的鸡堆型艾美尔球虫3-1E蛋白氨基酸一级结构为基础,采用DNAStar软件和生物信息学在线分析程序Prot Param、SOSUI、IEDB、SYFPEITHI等预测3-1E蛋白理化性质和二级结构,并分析其序列跨膜区、可溶性、亲水性、可及性、柔韧性参数以及抗原指数,预测B细胞表位和T细胞表位的可能区域。3-1E蛋白由170个氨基酸组成,分子式C818H1257N213O272S3,分子质量18.5234 ku,理论等电点为4.25,半衰期为10 h;无跨膜区均为膜外区,是一种可溶性蛋白。其二级结构中α螺旋占31.76%,β转角为12.35%。B细胞表位预测区域:44-49、65-67、86-87、111-116;分值较高的T细胞表位区域:52-60、94-102、97-105、154-162、157-165。运用生物信息学方法确定3-1E存在多个抗原表位,对进一步研究3-1E的抗原性和研发优势表位疫苗具有重要意义。

鸡堆形艾美球虫3-1E基因真核表达质粒的构建及其抗球虫免疫保护作用

应用反转录-聚合酶链式反应(RT-PCR)技术从堆形艾美球虫SH株子孢子中扩增3-1E基因片段,将其克隆入真核表达载体pcDNA3.1(+)中,构建重组表达质粒pcDNA3.1(+)-3-1E。质粒纯化后体外转染293T细胞,通过间接免疫荧光技术和免疫组织化学技术检测3-1E蛋白在转染细胞中的表达情况。将构建的重组质粒以及空载体质粒分别于14、21日龄分2次经腿部肌肉注射免疫SPF雏鸡,28日龄除非免疫非感染对照组外各组攻击性感染5×104个堆形艾美球虫孢子化卵囊,观察真核表达质粒对球虫感染鸡的免疫保护作用并探讨其免疫保护机理。结果显示,与载体对照组相比,质粒pcDNA3.1(+)-3-1E 2次免疫后可显著提高脾CD8+T淋巴细胞亚型数量,并具有一定的抗球虫免疫保护作用,可显著减少每克粪便的卵囊排出量,降低十二指肠病变记分,减少增重下降等。

堆形艾美球虫广东株3-1E基因在毕赤酵母中的表达

以重组质粒pGEM-3-1E为模板,扩增了序列两端分别含有EcoRⅠ和XbaⅠ酶切位点的堆形艾美球虫(Eimeria acervulina)广东株3-1E基因(长度为529 bp),将3-1E基因克隆至巴斯德毕赤酵母分泌型表达载体pPICZαC中,构建了酵母表达质粒pPICZαC-3-1E。转化毕赤酵母X-33得到含有3-1E基因的重组酵母,甲醇诱导产生的目的蛋白经SDS-PAGE分析和免疫印迹检测,表明毕赤酵母成功表达了3-1E基因。

(E)-3-(4-羟苯基)-1-(哌啶-1-基)丙-2-烯-1-酮衍生物核磁共振特征与构象分析(英文)

设计并合成了一系列(E)-3-(4-羟苯基)-1-(哌啶-1-基)丙-2-烯-1-酮衍生物.通过一维和二维核磁共振实验完成了1H和13C信号的指认.通过ROESY、变温实验和分子模拟技术对这类化合物的构象进行了分析.

鸡柔嫩艾美尔球虫ZJ株3-1E基因的原核表达及鉴定

本实验对E.tenellaZJ株的3-1E基因进行了克隆和表达。根据已报道的柔嫩艾美尔球虫3-1E基因序列设计引物,以孢子化卵囊总RNA为模板,用RT-PCR方法扩增得到一条特异片段,将扩增产物克隆至pUCM-T,转化感受态菌DH5α,经酶切鉴定获得阳性重组质粒并对其进行测序。测序结果与参考序列比较,核苷酸同源性为99.5%。然后将重组质粒和表达载体pET-30a分别以EcoRⅠ、SalⅠ酶切后构建重组表达载体pET-30a-3-1E,并将其转化入大肠杆菌BL21中,提取质粒经酶切和PCR鉴定正确后,用IPTG诱导表达。表达产物经SDS-PAGE和Westernblot检测显示,3-1E基因在大肠杆菌中成功表达;融合蛋白的分子量约为27ku,诱导6h的蛋白表达量可达到47.024%。

柔嫩艾美耳球虫杨凌株3-1E基因表达产物的初步免疫效果研究

用柔嫩艾美耳球虫(Eimeria tenella)子孢子表面抗原基因原核细胞表达产物免疫雏鸡,观察其对球虫攻击的免疫保护作用。将可溶性重组蛋白和包涵体重组蛋白经肌肉注射分别于7日龄、14日龄两次免疫罗曼小公雏,同时设攻虫对照组和空白对照组,于第2次免疫后1周(28日龄)用1×104个E.tenella卵囊进行攻毒,观察其诱导产生的保护力。结果表明,3-1E可溶性蛋白组免疫无论从卵囊计数、盲肠病变计分和相对增重均优于包涵体免疫组。说明3-1E基因在大肠杆菌中的表达产物对鸡柔嫩艾美耳球虫的攻击有一定的免疫保护作用。

MicroRNA-363-3p inhibits colorectal cancer progression by targeting interferon-induced transmembrane protein 1

BACKGROUND The molecular mechanisms of colorectal cancer development and progression are far from being elucidated.AIM To investigate the role of microRNA-363-3p(miR-363-3p)in the progression of colorectal cancer.METHODS Real-time polymerase chain reaction was performed to detect miRNA expression in human colorectal cancer tissues and paired normal colorectal tissues.PITA 6 was utilized to predict the targets of miR-363-3p.Dual-luciferase reporter system was used to validate the target of miR-363-3p.Plate colony formation assay and wound-healing assay were performed to evaluate cancer cells’clonogenic survival ability and migration ability,respectively.Cell proliferation was examined by cell counting kit-8 assay.Immunohistochemical staining was used to determine the expression level of interferon-induced transmembrane protein 1(IFITM1)in colorectal cancer tissues and adjacent tissues.The TCGA and GTEx databases were used to compare the expression levels of IFITM1 mRNA in colorectal cancer tissues and normal colorectal tissues and analyze the correlation between the expression levels of IFITM1 mRNA and overall survival and disease-free survival of patients.A colorectal cancer cell line with a deficiency of IFITM1 was constructed,and the regulation effect of IFITM1 on the clonogenic growth of colorectal cancer cells was clarified.RESULTS MiR-363-3p was decreased in colorectal cancer tissues compared to normal colorectal tissues.IFITM1 was characterized as a direct target of miR-363-3p.Overexpression of miR-363-3p led to decreased clonogenic survival,proliferation,and migration of colorectal cancer cells,which could be reversed by forced IFITM1 expression.CONCLUSION MiR-363-3p can constrain clonogenic survival,proliferation,and migration of colorectal cancer cells via targeting IFITM1.

柔嫩艾美耳球虫杨凌株3-1E基因的克隆、表达与蛋白结构预测

应用PCR技术,从柔嫩艾美耳球虫(Eimeria tenella)孢子化卵囊子孢子cDNA表达文库中扩增得到鸡E.tenella杨凌株(YL)子孢子表面抗原3-1E基因。序列分析表明,E.tenella YL 3-1E基因的开放阅读框(ORF)为513个碱基,编码170个氨基酸,与报道的E.tenella甘肃株(GS)3-1E基因相似性为99.8%,两者推导的氨基酸序列相似性为99.4%;而与文献报道的堆型艾美耳球虫(E.acervulina)美国株(US)3-1E基因序列的相似性为98.8%,推导的氨基酸序列相似性为98.8%。利用生物信息学和分子生物学软件对3-1E基因编码的蛋白进行结构预测,结果表明,该蛋白为结构松散的球状蛋白。将3-1E基因亚克隆到表达载体pGEX-4T-1,构建pGEX-3-1E重组质粒并在大肠杆菌BL21中进行表达,表达产物经SDS-PAGE分析,表明成功地表达出了分子量为44.7 ku的融合蛋白。该研究为球虫基因工程疫苗的研制奠定了基础。

堆型艾美球虫广东株3-1E基因的克隆与序列分析

根据GenBank中登录的堆型艾美球虫31E基因序列,设计了3条引物,以广东株堆型艾美球虫裂殖子总RNA为模板,利用反转录聚合酶链反应(RTPCR)扩增获得了31E基因部分片段,将这一片段克隆至pGEMTEasy载体中,经PCR、限制性内切酶分析和克隆片段的序列测定、比较,证实了克隆片段的可靠性。序列比较发现,所克隆的基因片段与Eimeriaacervulina美国株(US)、E.acervulinaQH株31EcDNA的核苷酸同源性分别为99.0%和99.2%,推导氨基酸的同源性分别为98.2%和97.6%。

鸡柔嫩艾美耳球虫3-1E真核重组表达质粒的构建及其免疫保护性分析

为构建鸡柔嫩艾美耳球虫(E.tenella)重组真核表达质粒并检测其免疫保护性,本实验将E.tenella表面抗原基因3-1E(E)与鸡IFN-γ、IL-2基因分别串联在真核表达载体pcDNA3.1(+)中,构建真核重组表达质粒pcDNA-E-IFNγ、pcDNA-E-IL2,同时构建对照质粒pcDNA-E、pcDNA-IFNγ、pcDNA-IL2。经酶切鉴定得到预期大小目的片段,测序结果与预期序列一致。将构建的重组质粒免疫鸡只,经western blot检测表明该重组质粒在鸡体内可以正常表达;对感染鸡只的免疫保护效果显示,pcDNA-E-IFNγ、pcDNA-E-IL2组抗球虫指数(ACI)分别为181.04、187.30,具有高效的抗球虫效果,为将其进一步研制成为具有实用价值的抗球虫疫苗奠定了基础。