Antibodies generated in response to plasmid DNA encoding zona pellucida glycoprotein-B inhibit in vitro human sperm-egg binding

To investigate the immunogenicity of plasmid DNA encoding bonnet monkey (Macaca radiata) zona pellucida (ZP) glycoprotein-B (bmZPB), the cDNA corresponding to bmZPB, excluding the N-terminal signal sequence and C-terminus transmembrane-like domain, was cloned in mammalian expression vector VR1020 downstream of tissue plasminogen activator signal sequence under cytomegalovirus promoter (VRbmZPB). In vitro transfection of COS-1, COS-7, CHO, HEK-293, and UM-449 mammalian cells with VRbmZPB plasmid DNA led to the expression of bmZPB. Expression of bmZPB in transfected cells was cytosolic. Flow cytometry analysis of COS-1 cells transfected with VRbmZPB revealed that approximately 15% cells expressed bmZPB. The expressed bmZPB has an apparent molecular weight of 57 kDa. Immunization of male BALB/cJ mice with VRbmZPB plasmid DNA in saline as compared to VR1020 immunized group, elicited significant antibodies against E. coli expressed recombinant bmZPB as evaluated in ELISA. The antibodies generated by VRbmZPB plasmid DNA recognized bonnet monkey as well as human ZP. The immune sera obtained from mice immunized with VRbmZPB plasmid DNA also inhibited, in vitro, the binding of spermatozoa to the ZP in the hemizona assay. These studies, for the first time, demonstrate the feasibility of DNA vaccine to generate antibodies against ZP that recognize native protein and inhibit human sperm-oocyte binding.     

人前胰岛素原基因裸质粒DNA肌肉注射可显著降低链脲佐菌素诱发糖尿病小鼠的血糖水平

通过基因治疗的方法补充胰岛素已用于实验性治疗胰岛素依赖型糖尿病(IDDM)。本研究构建了含有重组人前胰岛素原基因的棵质粒DNA载体(pCMV—IN),将其肌肉注射入链脲佐菌素(STZ)诱发的糖尿病C57小鼠体内,并辅以电穿孔方法,以获得在体胰岛素转基因治疗。该质粒载体表达的胰岛素mRNA,可通过RT—PCR方法在转基因局部的骨骼肌组织中检测到。在接受pCMV—IN注射的糖尿病小鼠中,血浆胰岛素水平显著升高,达到了未注射STZ的正常对照小鼠的水平,且胰岛素的表达可持续至少35d。pCMV—IN质粒注射转基因治疗显著降低了糖尿病小鼠在第7至35d的血糖水平,其下降幅度约6mmol／L;转基因治疗也显著降低了严重糖尿病小鼠的死亡率,其第6周时的死亡率由100％降为37％。结果表明,直接肌肉注射含人前胰岛素原基因裸质粒可获得胰岛素的有效表达,显著降低糖尿病小鼠的血糖水平并降低严重糖尿病小鼠的死亡率。裸质粒注射胰岛素转基因治疗有望成为IDDM的一种有效治疗手段。     

人呼吸道合胞病毒RNA聚合酶复合体蛋白N、P的表达鉴定

本研究根据编码A亚型人呼吸道合胞病毒(HumanRespiratorySyncytialVirus,HRSV)核壳体蛋白(Nucleocapsidprotein,N)和磷蛋白(phosphoprotein,P)的基因序列,各设计一对特异性的引物,应用RT-PCR技术,从感染HRSV的HEp-2细胞中扩增获得n和p的基因片断,克隆至真核表达载体pcDNA3.1( )。获得的重组质粒通过脂质体Lipofectamine2000转染COS-7细胞,72h后再用Westernblot鉴定蛋白的表达。结果显示真核表达载体pcDNA3.1( )/N和pcDNA3.1( )/P的限制性内切酶分析结果与预期一致,基因序列分析显示没有发生无义突变,利用蛋白印记方法也检测到了N和P的特异性条带。于是我们认为成功构建了含有HRSVN和P编码基因的真核表达载体,在真核细胞内能顺利表达。为进一步开展HRSV反向遗传学等研究奠定了基础。     

人巨细胞病毒DNA疫苗免疫效果的初步研究

人巨细胞病毒(HCMV)是广泛感染人类的重要病原体之一,在人群中有较高的感染率,胎儿、婴儿、器官移植及免疫缺陷者等更易感染.现有的抗HCMV药物治疗及被动免疫由于毒性大、价格昂贵及短效性等原因,应用大受限制,为此研制安全、有效的疫苗即成为防治HCMV疾病的重要手段.HCMV具有潜在的致癌作用,不宜发展活疫苗或含完整病毒DNA的死疫苗;亚单位疫苗虽然比较安全,但所用抗原不能在宿主细胞内表达,只能诱导体液免疫,这对于胞内感染的HCMV预防效果较差;通过新佐剂的应用,虽可引发细胞免疫,但免疫原性较差,且成本较高.因此,目前倾向于研制具有免疫原性的HCMV DNA疫苗.     

Construction of CpG motif-enriched DNA vaccine plasmids for enhanced early immune response

A DNA vaccine methodology using eukaryote expression vectors to produce immunizing proteins in the vaccinated hosts is a novel approach to the development of vaccine and immuno-therapeutics, and it has achieved considerable success over several infectious diseases and various cancers. To further enhance its efficiency, attempts were made to develop novel plasmid vectors containing multiple immunostimulatory CpG motifs, for rapid and strong immune response. First, a 2.9 kb compact plasmid vector (pVAC), containing CMV promoter, polycloning site, BGH poly(A) terminator, ampicillin resistance gene and pBR322 origin was constructed. A pVAC-hEPO was also constructed, which contained a human erythropoietin gene, for evaluating the transfection efficiency of naked plasmid DNA bothin vitro andin vivo. To examine the adjuvant effect of multi-CpG motifs on naked plasmid DNA, 22 and 44 enriched and unmethylated CpG motifs were introduced into pVAC to generate pVAC-ISS1 and pVAC-ISS2, respectively. 100 μg of pSecTagB, pVAC, pVAC-ISS1 or pVAC-ISS2 were each injected intramuscularly into the tibilias anterior muscle of Balb/c mice. The level of interleukin-6 induced in the mice injected with pVAC-ISS1 and pVAC-ISS2 were significantly elevated, after 12 hours, which were almost 2 and 2.5 times higher than that in the mice injected with pSecTagB, respectively. These results suggest that DNA vaccine plasmids with enriched CpG motifs can induce rapid secretion of interleukin-6 by lymphocytes. In conclusion, these vectors can contribute to the development of adjuvant-free DNA vaccinations against infectious diseases and various cancers.     

Enhancement of immunity and resistance in mice by pig IL-6 gene and CpG motifs encapsulated in chitosan nanoparticle

This study was conducted to explore the synergetic effect of a novel plasmid containing a porcine IL-6 gene and CpG motifs on immunity of mice in order to develop an effective adjuvant to boost resistance against infection. The synthetic oligodeoxynucleotide containing 11 CpG motifs was inserted into the reconstructed VR1020 plasmid containing the pig IL-6 gene (VRPIL6), designated VRIL6C, and then encapsulated in chitosan nanoparticles (CNP) prepared by ionic cross linkage, designated VRIL6C-CNP. The 3-week old mice were injected, respectively, with VRIL6C-CNP, VRIL6-CNP, CpG-CNP and VR1020-CNP to detect the changes of immunity. At 28 days post inoculation, the mice were challenged with virulent hemolytic serotype 2 Streptococcus to test their resistance against infection. The results showed that there was a significant increase in immunoglobulins and interleukins in mice receiving VRIL6C-CNP compared with the control groups, as well as an increase in the lymphocytes and monocytes in the inoculated mice, so that the immunity was remarkably improved in the VRIL6C-CNP group. The challenge provoked stronger immunity and protection against infection in the VRIL6C-CNP group than in the control mice that manifested severe symptoms and lesions. This suggests that VRIL6C-CNP could remarkably enhance the nonspecific immunity of mice, and facilitate the development of an effective immunopotentiator to promote the resistance of the animals against infection.     

Co-transfected SV40 origin of replication activates expression from SV40 promoterless constructs.

Co-transfection with expression plasmids is widely used to control DNA uptake efficiency in transient transfection experiments. However, a number of problems have been associated with their use. Here, we describe the activation of expression of constructs not containing the simian virus 40 (SV40) origin of replication (ori) by co-transfection in COS-7 cells with plasmids containing the SV40 ori. This effect has consequences for the use of such plasmids to control transfection efficiency.     

MPT63核酸疫苗的制备及其免疫原性

扩增结核分枝杆菌分泌蛋白MPT63编码序列 ,克隆于真核表达载体pJW 4 30 3中 .转染COS 7细胞 ,用Western印迹检测表明该基因在细胞内得到正确表达 .用该质粒连续免疫C5 7BL 6小鼠 3次后 ,用纯化的重组蛋白MPT63检测小鼠血清中的抗体 ,发现抗体滴度达到 10 5,免疫动物中γ干扰素的含量达到 2 5 8± 0 2U ml ,为空载体DNA免疫对照组的 2 0倍以上 ,说明MPT63核酸疫苗在实验动物体内引起了较强的免疫应答     

不同电转仪的电转参数、质粒用量和拓扑结构对猪胎儿成纤维细胞转染效率的影响

为获得猪胎儿成纤维细胞(porcine fetal fibroblasts, PFFs)最佳的电转染效率,本研究利用荧光激活细胞分选技术(fluorescence activated cell sorting, FACS)辅助优化NEPA 21和Nucleofector? 2b两种电转仪电转染PFFs细胞的参数,比较不同质粒用量和拓扑结构在ECM^? 830、NEPA 21和Nucleofector? 2b中的转染效率。结果显示：NEPA 21电转PFFs的最佳穿孔参数为脉冲电压200 V,脉冲长度3 ms,脉冲间隔50 ms,脉冲次数3次,脉冲电压衰减幅度10%;Nucleofector? 2b在U-023的转染参数下达到最高转染效率。ECM^? 830和Nucleofector? 2b的最适质粒用量都为10 μg,而NEPA 21为8 μg;超螺旋质粒比线性化质粒的转染效率更高,且3种仪器中Nucleofector? 2b转染效果最佳。本研究综合考虑电转仪、电转参数、质粒用量和拓扑结构的影响因素以优化PFFs的电转条件,为高效制备转基因猪及基因编辑猪的研究奠定基础。     

Lipid composition of cationic nanoliposomes implicate on transfection efficiency

Abstract

Cationic liposome (CL)-DNA complexes (lipoplexes) have appeared as leading nonviral gene carriers in worldwide gene therapy clinical trials. Arriving at therapeutic dosages requires the full understanding of the mechanism of transfection. However, using CLs to deliver therapeutic nucleic acids and drugs to target organs have some problems, including low transfection efficiency. The aim of this study was developing novel CLs containing four neutral lipids; cholesterol, 1,2-dioleoyl phosphatidylethanolamine, distearoylphosphatidylcholine and dipalmitoylphosphatidylcholine as a helper lipid and dimethyl dioctadecyl ammonium bromide as a cationic lipid to increase transfection efficiency. We have investigated the correlation between number of lipid composition and transfection efficiency. The morphology, size and zeta potential of liposomes and lipoplexes were measured and lipoplexes formation was monitored by gel retardation assay. Transfection efficiency was assessed using firefly luciferase reporter assay. It was found that transfection efficiency markedly depended on liposome to plasmid DNA (pDNA) weight ratio, lipid composition and efficiency of pDNA entrapment. High transfection efficiency of plasmid by four component lipoplexes was achieved. Moreover, lipoplexes showed lower transfection efficiency and less cytotoxicity compared to Lipofectamine?. These results suggest that lipid composition of nanoliposomes is an important factor in control of their physical properties and also yield of transfection.     

Effective naked plasmid DNA delivery into stem cells by microextrusion-based transient-transfection system for in situ cardiac repair

Background aims. Combining the use of transfection reagents and physical methods can markedly improve the efficiency of gene delivery; however, such methods often cause cell damage. Additionally, naked plasmids without any vector or physical stimulation are difficult to deliver into stem cells. In this study, we demonstrate a simple and rapid method to simultaneously facilitate efficient in situ naked gene delivery and form a bioactive hydrogel scaffold. Methods. Transfecting naked GATA binding protein 4 (GATA4) plasmids into human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) by co-extruding naked plasmids and hUC-MSCs with a biomimetic and negatively charged water-based biodegradable thermo-responsive polyurethane (PU) hydrogel through a microextrusion-based transient-transfection system can upregulate the other cardiac marker genes. Results. The PU hydrogels with optimized physicochemical properties (such as hard-soft segment composition, size, hardness and thermal gelation) induced GATA4-transfected hUC-MSCs to express the cardiac marker proteins and then differentiated into cardiomyocyte-like cells in 15 days. We further demonstrated that GATA4-transfected hUC-MSCs in PU hydrogel were capable of in situ revival of heart function in zebrafish in 30 days. Conclusions. Our results suggest that hUC-MSCs and naked plasmids encapsulated in PU hydrogels might represent a new strategy for in situ tissue therapy using the microextrusion-based transient-transfection system described here. This transfection system is simple, effective and safer than conventional technologies.     

小鼠白细胞介素33真核表达质粒的构建及表达

克隆小鼠IL-33基因构建其真核表达质粒,并转染COS-7细胞检测其表达。提取C57BL/6小鼠肺组织总RNA,经反转录聚合酶链式反应（RT-PCR）扩增小鼠IL-33基因,酶切后插入pcDNA^TM3.1/myc HisA构建其真核表达质粒pcDNA-3.1-IL-33,重组质粒转染COS-7细胞,RT-PCR和免疫印迹法（western blotting）检测目的基因表达。结果显示,pcDNA3.1-IL-33中插入的片段序列测定结果与小鼠IL-33cDNA序列一致,重组质粒转染COS-7细胞后检测到相应mRNA及蛋白表达。成功克隆了小鼠IL-33基因cDNA,并构建其真核表达质粒。     

VEGF重组质粒pcDNA/V的构建及其在大鼠急性心肌缺血模型中的表达

构建人血管内皮细胞生长因子(VEGF165)真核表达载体,并研究其在细胞水平和大鼠急性心肌梗死动物模型中的表达。利用RT-PCR方法,从人扁桃体组织中扩增人VEGF165基因,构建真核表达载体pcDNA/V。应用脂质体介导的基因转移技术,将pcDNA/V转染至人胚肾细胞(293细胞)中,经G418筛选获得稳定表达的重组质粒细胞克隆。ELISA、Westernblot检测证实重组质粒pcDNA/V能在293细胞中高效表达外源VEGF基因,鸡胚绒毛尿囊膜血管生成实验证实表达产物具有促血管生成的活性。进一步的体内表达研究,建立大鼠急性心肌梗死模型,将重组质粒pcDNA/V、空质粒pcDNA3.1( )分三点注射于梗死交界处心肌内,四周后取材。经免疫组化染色检测,pcDNA/V组在梗死交界区有VEGF阳性表达;电镜观察显示,pcDNA/V组在梗死交界处心肌细胞间有大量毛细血管内皮细胞增生。实验结果表明成功克隆了人VEGF165基因,构建了其真核表达载体。体内、外表达研究证实重组质粒的表达产物具有促血管生成的生物学活性,为VEGF基因治疗缺血性心肌病的研究提供实验基础。     

准噶尔小胸鳖甲抗冻蛋白MpAFP698在真核细胞中的表达与鉴定

目的:构建真核表达质粒pEGFP-N1-mpafp698,转染人胚肾293T细胞,并表达准噶尔小胸鳖甲抗冻蛋白MpAFP698.方法:PCR扩增出mpafp698序列,将其克隆入本室保存的真核表达载体pEGFP-N1中,转染293T细胞;利用RT-PCR、流式细胞仪、免疫荧光、Western 印迹检测蛋白表达.结果:构...     

NanoSMGT: transgene transmission into bovine embryos using halloysite clay nanotubes or nanopolymer to improve transfection efficiency

The objectives were to investigate whether: 1) nanotransfectants are more effective than other common transfection methods for SMGT; 2) NanoSMGT is able to transmit exogenous DNA molecules to bovine embryos; and 3) halloysite clay nanotubes (HCNs) can be used as a transfection reagent to improve transgene transmission. Four transfection systems were used: naked DNA (without transfectant), lipofection, nanopolymer, and halloysite clay nanotubes. Plasmid uptake by sperm and its transfer to embryos were quantified by conventional and real-time PCR, as well as EGFP expression by florescence microscopy. Furthermore, sperm motility and viability, and embryo development were investigated. Mean number of plasmids taken up was affected (P < 0.05) by transfection procedure, with the nanopolymer being the most effective transfectant (∼153 plasmids per spermatozoon). None of the treatments affected sperm motility or viability. The mean number of plasmids transmitted to four-cell stage embryos was higher (P < 0.05) in nanopolymer and HCNs than liposomes and naked DNA groups. The number of embryos carrying the transgene increased from 8–10% using naked DNA or liposomes to 40–45% using nanopolymer or HCN as transfectants (P < 0.05). There were no significant differences among transfection procedures regarding blastocyst formation rate of resulting embryos. However, no EGFP-expressing embryo was identified in any treatment. Therefore, nanotransfectants improved transgene transmission in bovine embryos without deleterious effects on embryo development. To our knowledge, this was the first time that bovine embryos carrying a transgene were produced by NanoSMGT.     

SV40 T基因转化的山羊乳腺上皮细胞系及其生物学特性

目的建立能用于乳腺特异表达基因构件质量检验的山羊乳腺上皮细胞系.方法根据已发表的SV40病毒T基因序列设计引物,以整合有SV40 DNA早期基因区的COS-1细胞基因组DNA为模板,用高保真PCR扩增SV40 T基因.将获得的SV40 T基因克隆入真核表达载体,并用获得的重组表达质粒转染山羊原代乳腺上皮细胞.经有限稀释和反复传代后获得转化细胞克隆,对其生物学特性进行研究.结果扩增出序列正确的SV40T基因,重组质粒转染获得的转化细胞的对数生长期为接种后第4天,细胞群体倍增时间为23.5*!h,克隆形成率为26.7%.DNA斑点杂交试验证明转化细胞的基因组中整合有SV40 T基因,染色体核型分析试验表明转化细胞的核型无明显异常,裸鼠接种试验证明转化细胞不能形成肿瘤,软琼脂集落形成试验表明转化细胞在软琼脂中不能生长.部分细胞克隆已在体外传30代以上,保持正常乳腺上皮细胞的形态特征,在胶原基质上能形成腺泡样结构.结论本研究获得的SV40 T基因转化的山羊乳腺上皮细胞具有转化细胞系的生物学特性.     

人CD46 RNAi 逆转录病毒载体系统的构建与鉴定

目的:构建并筛选携带针对CD46基因的pSUPER retro RNAi逆转录病毒载体,从而特异、有效地抑制人CD46mRNA水平。方法:利用DNA重组技术,将2条60nt能转录产生靶向CD46小发夹RNA(shRNA)的寡核苷酸序列,定向克隆入逆转录病毒载体pSUPER retro,并转化大肠杆菌JM109。结果:重组载体经PCR及限制性内切酶酶切鉴定初步成功后送测序,结果表明序列正确。结论:特异性沉默CD46基因的pSUPER retro RNAi逆转录病毒载体构建成功,为后续转染Jurkat细胞,研究CD46在T细胞的信号转导中的作用奠定了基础,对进一步研究T细胞相关疾病及开展细胞免疫缺陷的治疗方面提供了新的思路与方向。