血吸虫硫氧还蛋白免疫原性研究Ⅰ日本血吸虫硫氧还蛋白DNA疫苗的构建和鉴定

目的构建日本血吸虫(大陆株)硫氧还蛋白(Trx)DNA疫苗(pcDNA3-SjcTrx),并进行分子生物学鉴定。方法根据日本血吸虫菲律宾株Trx基因序列设计一对引物,上游引物引入BamHI酶切位点和起始密码子ATG,下游引物引入EcoRI酶切位点和终止密码子TCA。以日本血吸虫大陆株成虫总RNA为模板,经反转录-聚合酶链反应(RT-PCR)扩增日本血吸虫大陆株Trx(SjcTrx)编码基因。经双酶切并纯化的PCR产物与经双酶切纯化的pcDNA3质粒DNA片段用T4DNA连接酶连接,克隆入真核表达载体pcDNA3,构建重组质粒pcDNA3-SjcTrx,并经限制性内切酶双酶切、琼脂糖凝胶电泳、PCR检测和核苷酸序列测定进行鉴定。结果SjcTrx编码基因RT-PCR产物约334bp,构建的pcDNA3-SjcTrx重组质粒DNA经限制性内切酶双酶切和PCR扩增产物于琼脂糖凝胶电泳均观察到相同大小基因片段,根据核苷酸序列测定结果推导的氨基酸序列与日本血吸虫菲律宾株和曼氏血吸虫Trx分别有97%和43%的同源性。结论日本血吸虫(大陆株)硫氧还蛋白DNA疫苗构建成功,为开展动物保护性免疫试验创造了条件。     

日本血吸虫Calpain序列分析及DNA疫苗体系的构建

目的　探讨日本血吸虫疫苗候选分子 -钙离子激活的中性蛋白激酶 (Calpain)在抗日本血吸虫感染的保护性作用及其保护性免疫机制。方法　从日本血吸虫成虫中提取RNA ,用RT -PCR扩增Calpain含多个B ,T细胞表位的片段 ,引物中包含BamHI和EcoRI的酶切位点 ,PCR扩增产物经纯化后TA克隆 ,转化的阳性TA克隆经PCR筛选后 ,液体培养大肠杆菌并回收质粒DNA ,质粒DNA经BamHⅠ和EcoRⅠ双酶切 ,目的基因亚克隆到真核表达质粒pVAC载体 ,构建 pVAC -Cal pain真核表达体系 ,转化的阳性亚克隆经PCR筛选 ,液体培养大肠杆菌并回收pVAC -Calpain质粒DNA ,质粒DNA经BamHⅠ和EcoRⅠ双酶切和DNA序列分析鉴定被亚克隆的Calpain基因。 结果　RT -PCR从日本血吸虫RNA中扩增了 4 5 3bp的Calpain基因 ,经BamHⅠ和EcoRⅠ双酶切和DNA序列分析鉴定Calpain基因被克隆到真核表达质粒 pVAC载体。 结论　日本血吸虫疫苗候选分子Calpain的DNA疫苗体系的建立将有助于解析这个疫苗候选分子抗日本血吸虫感染的保护性免疫作用及保护性免疫机制。     

日本血吸虫26kDa谷胱甘肽S-转移酶基因真核表达载体的构建及序列测定

目的 扩增日本血吸虫２６ｋＤａ谷胱甘肽Ｓ－转移酶（Ｓｊ２６ＧＳＴ）基因片段，构建其重组真核表达载体，以研制ＳｊＧＳＴ核酸疫苗。方法 根据已知基因序列设计一对引物，运用ＲＴ－ＰＣＲ技术扩增Ｓｊ２６ＧＳＴ目的基因片段，将其克隆到ｐｃＤＮＡ３质粒中，并经酶切、ＰＣＲ鉴定，测序证实。结果 获得ＧＳＴ－ｐｃＤＮＡ３重组克隆。结论：本实验获得Ｓｊ２６ＧＳＴ重组克隆，为进一步研制核酸疫苗创造了条件。     

编码日本血吸虫线粒体大亚基核糖体基因的亚克隆、测序及同源性分析

目的筛选日本血吸虫成虫 cDNA 文库,得到基因克隆并测序。方法体外将以阳性克隆为模板的 PCR 产物和 pGEM-T 载体连接,转染大肠杆菌 XL1-blue,经抗生素及生色底物 X-gal 初筛,再用限制性内切酶酶切法进一步鉴定为重组质粒后,DNA 自动测序仪测序。序列送blast 基因服务站进行同源性分析。结果构建三个含日本血吸虫 cDNA 基因片段的重组子,其中一个阳性克隆序列为编码日本血吸虫线粒体大亚基核糖体的基因序列。结论获得编码日本血吸虫线粒体大亚基核糖基因片段,为分析其作为候选重组疫苗分子的潜能打下基础。     

日本血吸虫脂肪酸结合蛋白DNA疫苗诱导小鼠保护性免疫力的研究

目的构建日本血吸虫脂肪酸结合蛋白(Sj14FABP)重组质粒,并观察其在小鼠体内诱导的抗日本血吸虫感染保护作用。方法RT-PCR特异性扩增Sj14FABP基因,将其克隆入真核表达载体pVIVO2,通过PCR、双酶切及测序鉴定。获得的重组质粒pVIVO2-Sj14FABP转染HepG2细胞,间接免疫荧光(IFA)检测蛋白表达;并免疫BALB/c小鼠,30d后攻击感染,感染后45天剖杀,计数检获成虫数和肝脏虫卵数。结果RT-PCR扩增出大小为440bp的Sj14FABP片段,重组质粒经PCR和双酶切后均获得目的片段,转染细胞IFA结果显示有较强荧光。重组质粒免疫小鼠后分别获得24.1%减虫率和27.2%减卵率。结论成功构建和表达重组质粒pVIVO2-Sj14FABP,该核酸疫苗能够诱导小鼠产生部分抗血吸虫感染的保护力。     

日本血吸虫抗原表位编码基因重组质粒的高效构建与鉴定

目的探索用一种新方法—胶内连接法,快速构建日本血吸虫抗原表位编码基因重组表达载体,高效克隆及表达日本血吸虫抗原表位,用于疫苗候选分子的筛选。方法将pUMCV质粒载体用SalI和EcoRI双酶切,酶切产物于低熔点胶中电泳,紫外灯下割取含酶切载体的条带。取一定量割取的低熔点胶,直接将其与合成的单个抗原表位编码基因或者是两个不同抗原表位编码基因进行连接。连接产物转化感受态菌DH5α。挑选阳性克隆进行双酶切、电泳及测序鉴定。结果单个抗原表位或两个不同的表位编码基因被成功地插入真核表达载体。结论采用胶内连接法直接在低熔点胶存在的条件下进行插入基因片段与质粒载体的连接,快速高效地获得了多个单一或组合抗原表位编码基因重组质粒,为进一步筛选日本血吸虫疫苗分子奠定了基础。     

日本血吸虫生殖相关抗原31～32kDa分子的质谱分析与鉴定

目的利用蛋白质组学技术分离、鉴定日本血吸虫天然分子31～32kDa抗原组分。方法收集感染后32d的日本血吸虫成虫，制备成虫蛋白，经一维电泳、固相pH梯度双向凝胶电泳分离，选取31-32kDa附近的蛋白点进行质谱分析鉴定，通过凝胶图像分析软件进行结果比较。结果二维电泳结果显示31～32kDa附近有13个蛋白点，经MALDI-TOF-MS鉴定及分析，发现3个蛋白分子分别与曼氏血吸虫Actine-2、日本血吸虫3-磷酸甘油脱氢酶、曼氏血吸虫组织蛋白酶B肽链内切酶有高度的同源性。结论31-32kDa组分中含有这3个蛋白点，它们与日本血吸虫31～32kDa天然分子中起保护性的有效分子密切相关。同时，它们的发现也为天然分子疫苗向基因工程疫苗的推进奠定了基础。     

日本血吸虫HGPRT编码基因的克隆与表达

目的 克隆和表达日本血吸虫大陆株次黄嘌呤鸟嘌呤磷酸核糖转移酶(HGPRT)编码基因。方法依据GenBank日本血吸虫HGPRT开放阅读框(ORF)设计一对引物，上游和下游引物分别引入BamH I和Sal I酶切位点。以日本血吸虫大陆株（安徽株，简称Sjc-A）成虫总RNA为模板，反转录PCR (RT-PCR)扩增日本血吸虫大陆株HGPRT(SjcHGPRT)全长编码基因。经双酶切纯化的PCR产物与同样双酶切纯化的pET28a质粒DNA片段用T4 DNA连接酶连接，构建重组质粒pET28a-SjcHGPRT，转化感受态E．coli BL21，并大量扩增。重组质粒DNA经限制性内切酶双酶切、PCR、琼脂糖凝胶电泳和核苷酸序列测定进行鉴定。pET28a-SjcHGPRT/E．coli BL21I程菌用IPTG诱导表达，重组蛋白用SDS-PAGE和Western blot分析。结果 SjcHGPRT编码基因RT-PCR产物约700 bp，构建的pET28a-SjcHGPRT重组质粒DNA经限制性内切酶双酶切和PCR扩增产物于琼脂糖凝胶电泳均观察到相同大小的基因片段，根据核苷酸序列测序结果推导的氨基酸序列与报道的日本血吸虫大陆株（湖南株，简称Sjc-H）及曼氏血吸虫HGPRT分别有99%和83%的同源性。获得的重组蛋白(reSjcHGPRT)经SDS-PAGE和Western blot分析，分子量约30 kDa，并能被抗His-G-HRP抗体、日本血吸虫感染小鼠血清和日本血吸虫病人血清识别。结论 日本血吸虫大陆株(安徽株)HGPRT表达获得成功，并获得了纯化重组蛋白，为开展该分子功能和免疫原性研究奠定了基础。     

中国大陆三个地区日本血吸虫基因组DNA的RFLP的初步研究

本文用三种限制性内切酶(HaeⅢ,EocRⅠ,HindⅢ)分析了中国大陆的四川西昌、安徽贵池和湖南君山三个地区的日本血吸虫基因组DNA的限制性片段长度差异(RFLP)。构建了三个地区日本血吸虫两种内切酶的RFLP的图谱。HaeⅢ和EcoRⅠ内切后经电泳检出四川地区日本血吸虫与湖南、安徽两地的日本血吸虫DNA重复序列的差异。其中HaeⅢ酶切图谱中有差异的片段为20.0kb、6.5kb、5.3kb、4.3kb和0.6kb;EcoRⅠ的图谱中为3.3kb、1.2kb和0.9kb。本实验结果从DNA分子水平为中国大陆日本血吸虫地域株分类问题提供了依据。     

日本血吸虫疫苗的研究进展与展望

血吸虫疫苗的研究已经纳入了WHO和我国主要疾病防治规划,并且取得了很大的进展.血吸虫疫苗的研究历史经历了从死疫苗、致弱活疫苗、亚单仲疫苗、基因工程疫苗、核酸疫苗到多价联合疫苗等的探索过程.近年来开展的血吸虫免疫机制和血吸虫基因组的研究对血吸虫疫苗的研制起到了积极的推动作用.该文主要对日本血吸虫疫苗近十年的研究进展作一综述.     

Sjcb2 DNA疫苗在血吸虫感染小鼠中的保护性作用研究

目的 研究Sjcb2 DNA疫苗在日本血吸虫病小鼠模型中的保护性作用和机制,为血吸虫疫苗的研究提供有效的候选抗原分子。方法 构建pcDNA3.1(+)/Sjcb2 核酸疫苗,将6周龄雌性BALB/c小鼠随机分为pcDNA3.1(+)/Sjcb2 核酸疫苗组、pcDNA3.1(+)空质粒组及生理盐水组,每组35只,采用后腿股四头肌注射方法,每次免疫质粒DNA 100 μg,每2周免疫1次,共免疫3次,用日本血吸虫尾蚴攻击感染各组小鼠。PCR及免疫组化法检测Sjcb2基因在小鼠体内的稳定性及表达情况;MTT法检测小鼠脾淋巴细胞特异性增殖反应;ELISA法检测小鼠血清中Sjcb2抗体水平及攻击感染前后脾淋巴细胞培养上清中IFN-γ和IL-4的水平;计数小鼠荷成虫对数和肝脏荷虫卵数。结果 疫苗组小鼠均可在小鼠肌细胞中检测到Sjcb2基因及其抗原的表达;DNA疫苗组T细胞增殖显著增高(P <0.05);ELISA 结果显示疫苗组IFN-γ 水平显著增高(P <0.05),血吸虫尾蚴攻击感染后各组小鼠IL-4水平显著升高(P <0.05)。DNA疫苗组小鼠荷成虫对数及肝脏荷虫卵数与其它组比较显著性减少(P <0.05),其减虫率为36.32％,减卵率为60.61％。结论 Sjcb2 DNA疫苗接种小鼠后能在小鼠肌细胞中稳定存在和表达;Sjcb2可能通过提高IFN-γ 和降低IL-4水平调节Th1细胞亚群产生抗血吸虫感染的保护性作用。     

Methylation of integrated adenovirus type 12 DNA sequences in transformed cells is inversely correlated with viral gene expression.

The adenovirus type 12 (Ad12) DNA sequences integrated into the DNA of four lines of Ad12-transformed hamster cells are extensively methylated. Methylation in mammalian cell DNA is believed to occur predominantly at 5'-C-G-3' sequences. The majority, although not all, of the 5'-C-C-G-G-3' sequences present in integrated Ad12 DNA are methylated. Ad12 DNA isolated from purified virions, on the other hand, is not methylated to any significant extent. The segments of the integrated viral DNA comprising early genes, which are expressed as mRNA in two lines of Ad2-transformed hamster cells, are undermethylated in comparison to late viral segments, which are not expressed and are extensively methylated. In contrast, in two lines of Ad12-induced rat brain tumor cells, some of the late viral genes have been shown to be expressed as mRNA. The segment of the integrated Ad12 DNA that comprises these late genes, the EcoRI B fragment, is undermethylated in comparison to the extensive methylation of the same fragment in Ad12-transformed hamster cells. Thus, there appears to exist a striking inverse correlation between the levels of methylation of specific DNA segments and the extent to which these segments are expressed as mRNA. The functional significance of this correlation remains to be determined. It may provide a clue to understanding the regulation of gene expression in transformed cells and perhaps in eukaryotic cells in general.     

Human tumor-derived genomic DNA transduced into a recipient cell induces tumor-specific immune responses ex vivo

This article describes a DNA-based vaccination strategy evaluated ex vivo with human cells. The vaccine was prepared by transferring tumor-derived genomic DNA to PCI-13 cells, a highly immunogenic tumor cell line ("recipient cell"), which had been genetically modified to secrete IL-2 (PCI-13/IL-2). PCI-13 cells expressed class I MHC determinants (HLA-A2) shared with the tumor from which the DNA was obtained as well as allogeneic determinants. DNA from a gp100(+) melanoma cell line was transduced into gp100(-) PCI-13/IL-2 cells (PCI-13/IL-2/DNA). A T cell line specific for the gp100 epitope responded to PCI-13/IL-2/DNA cells by IFN-gamma-secretion measured in enzyme-linked immunospot assays. The T cell line also recognized the gp100 epitope presented by dendritic cells that ingested PCI-13/IL-2/DNA cells, which had been induced by UVB irradiation to undergo apoptosis. After up-take and processing of apoptotic PCI-13/IL-2/DNA cells, the dendritic cells primed normal peripheral blood lymphocytes to generate effector T cells specific for the tumor donating the DNA. The results indicate that tumor epitopes encoded in such DNA are expressed in recipient cells and can induce tumor-specific T cells. The findings support translation of this vaccination strategy to a phase I trial in patients with cancer.     

乙型肝炎病毒e抗原结合蛋白2原核表达载体的构建及诱导表达

目的体外构建乙型肝炎病毒e抗原结合蛋白2原核表达载体并观察其表达情况。方法以含有HBEBP2全长序列的质粒为模板，通过PCR扩增获得HBEBP2基因片段，将HBEBP2连接到pGEM-T载体，在测序证实正确后将其插入至原核表达载体pET-32a（＋）中，转化BL21大肠埃希菌，经IPTG诱导，并通过SDS-PAGE和Westernblot分析鉴定融合蛋白的表达。结果扩增获得的HBEBP2基因片断被成功构建到大肠埃希菌原核表达载体中。经IPTG诱导，得到了融合蛋白的表达，经Westernblot分析证实其分子量为34kD，具有抗原性。结论体外成功表达HBEBP2蛋白，为进一步研究HBEBP2蛋白的免疫原性和生物学特性奠定了基础。     

幽门螺杆菌靶向核酸疫苗的构建及免疫学评价

目的:为了提高幽门螺杆菌DNA疫苗的免疫原性,构建具有靶向作用于APC细胞的核酸疫苗.方法:利用基因工程技术,将靶向基因片段和过氧化氢酶基因融合构建了核酸疫苗.体外转染293T细胞,间接免疫荧光和ELISA检测其基因表达情况.用该核酸疫苗肌肉免疫注射BALB/c小鼠后,测定其血清IgG、IgG1和IgG2a水平.结果:间接免疫荧光检测转染DNA疫苗的293T细胞,表明该疫苗能在真核细胞中表达目的抗原,通过ELISA方法测定表明其仍具有IgG抗体结合能力.BALB/c小鼠免疫后血清测定结果表明,该靶向核酸疫苗诱导的抗体水平高于对照质粒pDNAkat,抗体分型实验显示靶向核酸疫苗pcDNAkathIgz和pcDNAkat,促进了免疫反应类型由Th2向Th1的部分偏转.结论:成功构建了靶向APC细胞的幽门螺杆菌核酸疫苗,并在小鼠体内诱导了较高的抗体水平.     

DNA rearrangements in MPC-11 immunoglobulin heavy chain class-switch variants.

Immunoglobulin heavy chain class switching has been observed in vitro. In the IgG2b-producing MPC-11 mouse myeloma cell line, IgG2a-producing cells arise at a high frequency. In some cases, switch variants producing normal-sized (Mr 55,000) gamma 2a heavy chains have arisen spontaneously from a mutagen-induced "intermediate" (ICR 9.7.1) that produces an unusually large (Mr 75,000) heavy chain. Other switch variants have been isolated directly from the parent cell line. The expressed and unexpressed gamma 2b genes of MPC-11 can be distinguished in restriction endonuclease digests of total genomic DNA so that DNA rearrangements detected in MPC-11 variants can be directly associated with one or the other of these two genes. We describe here DNA rearrangements occurring on the expressed heavy chain chromosome of several MPC-11 gamma 2a switch variants and on the expressed chromosome of the ICR 9.7.1 intermediate. Our data indicate that all of these variants express the parental heavy chain variable region (VH) gene, supporting previous protein studies. We provide mapping data for the expressed gene of both ICR 9.7.1 and one of the IgG2a-producing variant cell lines (ICR 9.9.2.1) derived from it and discuss the advantages of an in vitro switching system for examining the dynamics of the immunoglobulin heavy chain class switch.     

Chromosomal telomere attrition as a mechanism for the increased risk of epithelial cancers and senescent phenotypes in type 2 diabetes

Telomeres are the repeat DNA sequences at the end of chromosomes necessary for successful DNA replication and chromosomal integrity. Telomeres shorten at cell division at a rate determined by oxidative DNA damage, and cells are triggered into replicative senescence once telomeres shorten to a critical length. Telomere-related chromosomal maintenance also has a role in carcinogenesis. Type 2 diabetes is characterised by increased oxidative stress, increased oxidative DNA damage, senescent retinal and renal phenotypes, and an increased risk of epithelial malignancy. We suggest that increased oxidative DNA damage and telomere attrition in type 2 diabetes leads to: (1) carcinogenic telomere-dependent chromosomal non-reciprocal translocations, genomic instability, and the development of epithelial cancers; (2) senescent retinal and renal phenotypes (expressed as diabetic retinopathy and nephropathy); and (3) senescent vascular endothelial, monocyte-macrophage and vascular smooth muscle cells (expressed as endothelial dysfunction and accelerated atherogenesis). An adverse intrauterine environment leads to increased feto-placental oxidative stress and feto-placental oxidative DNA damage. We also suggest that intrauterine oxidative DNA damage and telomere shortening is another point at which increased oxidative stress could contribute to a pre-programmed increased risk of senescent phenotypes in adult offspring, characterised by type 2 diabetes and epithelial malignancy. These suggestions can be used to understand early glucose intolerance in the young children of type 1 diabetes pregnancies, poor cancer outcomes in type 2 diabetes, beta cell fatigue in type 2 diabetes and the absence of increased epithelial cancer risk in type 1 diabetes.     

DNA/MVA vaccine for HIV type 1: effects of codon-optimization and the expression of aggregates or virus-like particles on the immunogenicity of the DNA prime

Recently, a vaccine consisting of DNA priming followed by boosting with modified vaccinia Ankara (MVA) has provided long-term protection of rhesus macaques against a virulent challenge with a chimera of simian and human immunodeficiency viruses. Here, we report studies on the development of the DNA component for a DNA/MVA HIV vaccine for humans. Specifically, we assess the ability of a codon-optimized Gag-expressing DNA and two noncodon-optimized Gag-Pol-Env-expressing DNAs to prime the MVA booster dose. The codon-optimized DNA expressed virus-like particles (VLPs), whereas one of the noncodon-optimized DNAs expressed VLPs and the other expressed aggregates of HIV proteins. The MVA boost expressed Gag-Pol and Env and produced VLPs. Immunogenicity studies in macaques used one intramuscular prime with 600 microg of DNA and two intramuscular boosts with 1 x 10(8) pfu of MVA at weeks 8 and 30. The codon-optimized and noncodon-optimized DNAs proved similar in their ability to prime anti-Gag T cell responses. The aggregate and VLP-expressing Gag-Pol-Env DNAs also showed no significant differences in their ability to prime anti-Env Ab responses. The second MVA booster dose did not increase the peak CD4 and CD8 T cell responses, but increased anti-Env Ab titers by 40- to 90-fold. MVA-only immunizations elicited 10-100 times lower frequencies of T cells and 2-4 lower titers of anti-Env Ab than the Gag-Pol-Env DNA/MVA immunizations. Based on the breadth of the T cell response and a trend toward higher titers of anti-Env Ab, we are moving forward with human trials of the noncodon-optimized VLP-expressing DNA.     

Effect of vector-expressed shRNAs on hTERT expression

AIM: To study the effect of short hairpin RNAs (shRNAs) expressed from DNA vector on hTERT expression. METHODS: Oligonucleotides coding for four shRNAs against hTERT were cloned into a mammalian shRNA expression vector pUC18U6 to form pUC18U6ht1-4, which were then introduced into HepG2 cells by using liposome-mediated transfection. HepG2 cells transfected by pUC18U6 and pUC18U6GFPsir, which expressed shRNA against green fluorescent protein (GFP), were used as controls. hTERT mRNA in the transfected cells were quantified by using real-time fluorescent RT-PCR. RESULTS: Among the four shRNAs against hTERT, two decreased the hTERT mRNA level. Compared with the controls, pUC18U6ht which expressed the two shRNAs reduced hTERT mRNA by 39% and 49% (P<0.05). CONCLUSION: hTERT expression is inhibited by the shRNAs expressed from the DNA vector.