小鼠IL-27真核表达载体的构建及其在RAW264.7 细胞中的表达

目的:构建小鼠白介素27(Interleukin 27,IL-27)单链融合基因的真核表达载体并检验其在RAW264.7细胞中的表达情况。方法:提取小鼠脾细胞总RNA,通过RT-PCR扩增出小鼠EBI3和p28 c DNA。采用重叠延伸PCR(splicing by overlap extension PCR,SOE PCR)通过编码疏水性多肽接头(Gly4Ser)3的DNA序列连接小鼠EBI3和p28基因片段,构建小鼠IL-27单链融合基因(mouse single chain IL-27,msc IL-27),并将其克隆至pc DNA3.1(+)载体。通过酶切和测序鉴定阳性重组载体,将重组质粒pc DNA3.1-IL-27通过脂质体转染法转染小鼠巨噬细胞株RAW264.7,通过RT-PCR方法检测目的基因的表达。结果:测序分析表明,小鼠IL-27单链融合基因中EBI3、linker和p28的连接顺序、方向及碱基序列与预期相符。在转染后的RAW264.7细胞中检测到了小鼠IL-27 m RNA的表达。结论:成功构建了小鼠IL-27单链融合基因及其真核表达载体,并在RAW264.7细胞中实现表达,为进一步探讨IL-27的生物学功能奠定了基础。     

小鼠IL-39的克隆及真核表达载体的构建

[目的]构建小鼠白介素(interleukin,IL)-39单链融合基因及其真核表达载体。[方法]通过RT-PCR得到小鼠EB病毒诱导基因3(Epstein-Barr virus-induced gene 3,EBI3)及IL-23p19基因的全长编码区。通过重叠延伸PCR和编码疏水性多肽接头(linker)(Gly4Ser)3的DNA序列将小鼠EBI3全长编码区及IL-23p19成熟肽编码区连接起来,构建小鼠IL-39单链融合基因,并将其克隆至真核表达载体pc DNA3.1/V5-His中,通过限制性内切酶双酶切及基因测序鉴定阳性重组载体。[结果]通过重叠延伸PCR得到1 254 bp大小的目的条带,测序分析显示,小鼠IL-39单链融合基因中EBI3、linker和IL-23p19的基因序列及连接顺序和方向均完全正确。[结论]成功构建了小鼠IL-39单链融合基因及其真核表达载体。     

细菌内同源重组法制备mIL-12腺病毒载体及其体外高效表达

利用细菌内同源重组法构建含 m IL- 1 2双顺反子重组腺病毒载体 ,其中由 polio IRES连接的 m IL- 1 2 p40和 p35双亚基目的基因片段用 Not +Xho 酶自真核表达载体 pc DNA3/m IL- 1 2中切出 ,亚克隆至经同样酶切的腺病毒穿梭质粒中 ,形成转移质粒 p Adtrack- CMV/m IL- 1 2 ,对之线性化后与腺病毒基因组质粒 p Adeasy- 1共转化 BJ51 83菌 ,抽提经鉴定含目的基因的重组体质粒DNA,转染 2 93细胞 ,包装成重组体腺病毒 Ad- m IL - 1 2 . Southern杂交证实 ,p40和 p35基因已被克隆至 Ad- m IL - 1 2基因组中 ;RT- PCR结果显示 ,Ad- m IL- 1 2感染的 MM45T· Li肿瘤细胞中有p40和 p35基因的表达 ,ELISA检测感染 48h细胞的上清中 m IL- 1 2的含量达 88ng每 1 0 6细胞 ,其体外能刺激小鼠脾脏淋巴细胞的增殖 ,提高 NK细胞活性及诱导 干扰素的产生 .结果表明 ,利用细菌内同源重组法制备的 Ad- m IL- 1 2重组腺病毒载体是一种方便、高效、成功率高的方法 ,所制备的重组体腺毒在体外能有效表达具有生物活性的 m IL- 1 2 ,为今后的体内应用奠定了基础 .     

鸡PPARγ基因转录本3上游开放阅读框转录后的调控作用

过氧化物酶体增殖物激活受体γ(peroxisome proliferator-activated receptor gamma,PPARγ)是脂肪生成的关键调控因子。本实验室前期研究发现,与人和鼠等哺乳动物PPARγ基因的转录本不同,鸡PPARγ基因的多个转录本5′UTR区存在上游开放阅读框(upstream open reading frames,u ORFs)。为了揭示该u ORF转录后的调控作用,本研究构建了鸡PPARγ基因转录本3(c PPARγ3)野生型5′UTR报告基因载体psi CHECK2-c PPARγ3-5′UTR-WT和u ORF突变(u ATG突变为终止密码子TGA)的5′UTR报告基因载体psi CHECK2-c PPARγ3-5′UTR-Mut。将这两个报告基因载体分别转染永生化鸡前脂肪细胞(immortalized chicken pre-adipocytes,ICPA)和鸡胚成纤维细胞DF1,检测海肾荧光素酶报告基因h Rluc活性及其m RNA表达。荧光素酶报告基因检测结果显示,在ICPA细胞中,psi CHECK2-c PPARγ3-5′UTR-Mut的h Rluc报告基因活性极显著高于psi CHECK2-c PPARγ3-5′UTR-WT(P0.01);在DF1细胞中,psi CHECK2-c PPARγ3-5′UTR-Mut的h Rluc报告基因活性高于psi CHECK2-c PPARγ3-5′UTR-WT,但差异不显著(P0.05)。q RT-PCR检测h Rluc基因m RNA表达结果显示,与psi CHECK2-c PPARγ3-5′UTR-WT相比,在ICPA细胞中,psi CHECK2-c PPARγ3-5′UTR-Mut转染细胞的h Rluc基因的m RNA表达水平极显著降低(P0.01);在DF1细胞中,psi CHECK2-c PPARγ3-5′UTR-Mut转染细胞后,h Rluc基因的m RNA表达水平也降低,但差异不显著(P0.05)。为进一步分析该u ORF对鸡c PPARγ3的转录后调控作用,本研究又分别构建了野生型c PPARγ3真核表达载体pc DNA3.1-c PPARγ3-WT和u ORF突变的c PPARγ3真核表达载体pc DNA3.1-c PPARγ3-Mut。q RT-PCR检测c PPARγ3的m RNA表达水平,结果显示,在这两种细胞中,pc DNA3.1-c PPARγ3-Mut转染细胞的c PPARγ3 m RNA表达水平均显著低于pc DNA3.1-c PPARγ3-WT转染细胞(P0.05),但Western blot结果显示,pc DNA3.1-c PPARγ3-Mut转染细胞的PPARγ蛋白表达水平极显著高于pc DNA3.1-c PPARγ3-WT转染细胞(P0.01)。这些研究结果表明,5′UTR区的u ORF抑制鸡c PPARγ3的翻译。     

脂质体介导含IL-2及IFN-γ cDNA腺相关病毒质粒的转基因及抗肿瘤作用

h IL- 2基因和 m IFN- γ基因经 IRES连接后克隆入腺相关病毒质粒表达载体 p AC中 ,构建得双基因质粒表达载体 p AC- FRI.体外经阳离子脂质体 Dosper介导转染小鼠肝癌细胞 MM45T.Li,Northern印迹及生物活性检测分别从 RNA水平和蛋白质水平证明了 2个基因的表达 .直接瘤内注射 Dosper- DNA复合物后 ,与对照组 ( Lac Z)相比 ,双基因组及 IL- 2或 IFN-γ单基因组均产生了较明显的抗瘤作用 ,并诱发了较高的特异 CTL活性 .     

人TIMP-1 cDNA克隆、真核表达载体构建及在COS-7细胞中的表达

根据 Gen Bank中 TIMP- 1基因的碱基序列 ,用 RT- PCR方法从人的正常肾组织中克隆出包含信号肽在内的 TIMP- 1全长 c DNA序列 .采用 T- A克隆的方法将之插入 p CRR2 .1中间载体 ,DNA测序证实该片段序列与文献报告的完全一致 .利用亚克隆的方法将 TIMP- 1 c DNA片段克隆到 pc DNA3载体上 ,构建出 pc DNA3/ TIMP- 1的真核表达载体 ,通过脂质体 DOTAP转染至 COS-7细胞 ,Northern印迹及原位杂交证实在 COS- 7细胞上获得人 TIMP- 1的高效表达 ,细胞增殖实验表明 TIMP- 1的高产表达可促进 COS- 7细胞的增殖 ,证实了所转染人 TIMP- 1的生物活性     

Lnc-RAP3对小鼠3T3-L1前脂肪细胞分化的影响

长链非编码RNA(long non-coding RNA,lnc RNA)是一类长度大于200nt、没有长开放阅读框架但往往具有m RNA结构特征的RNA,可以在转录及转录后水平参与基因的表达调控。近年来,有研究证实lnc RNA对脂肪生成具有重要作用。Lnc-RAP3位于小鼠(Mus musculus)17号染色体,其表达量在小鼠脂肪细胞分化前后呈现显著差异,但其具体的生物学功能尚不清楚。为探讨lnc-RAP3在小鼠3T3-L1前脂肪细胞成脂分化中的作用,本文首先构建了lnc-RAP3的真核表达载体pc DNA3.1-RAP3,利用脂质体将pc DNA3.1-RAP3和人工合成的lnc-RAP3的si RNAs分别转染3T3-L1前脂肪细胞,并对转染后的细胞进行诱导分化,并通过油红O染色、q RT-PCR检测成脂分化相关基因表达等方法比较过表达和敲降lnc-RAP3对3T3-L1前脂肪细胞成脂分化的影响。结果显示,过表达lnc-RAP3后,细胞内脂滴聚集显著减少(P0.05),在诱导分化第0 d、2 d和4 d时C/EBPα、Glut4、PPARγ、LPL和FAS的表达水平均呈显著(P0.05)或极显著(P0.01)下降;敲降lnc-RAP3后,细胞内脂滴聚集显著增多(P0.05),同时在诱导分化第0 d、2 d时PPARγ、LPL、C/EBPα、FAS和Glut4的表达水平呈显著(P0.05)或极显著(P0.01)升高。本研究结果表明,lnc-RAP3可能通过影响成脂分化相关基因的表达来抑制3T3-L1前脂肪细胞的成脂分化。     

小鼠GP73蛋白真核表达质粒的构建及其对mTOR转录水平的影响

目的:构建小鼠高尔基蛋白73(m GP73)的真核表达质粒,转染HEK293T细胞株验证重组质粒活性,并检测m GP73过表达情况下对小鼠肝癌细胞中mTOR m RNA水平的影响。方法:以实验室保存的H22小鼠肝癌细胞c DNA文库为模板,采用PCR技术扩增获得m GP73序列,将其插入pc DNA3.1-Flag-vector载体构建成重组质粒,转染HEK293T细胞后用蛋白免疫印迹检测融合蛋白的表达,并用q PCR检测在GP73过表达情况下H22细胞中mTOR转录水平的变化。结果:菌液PCR、重组质粒的双酶切结果及测序均表明重组质粒构建成功,蛋白免疫印迹表明m GP73蛋白在HEK293T细胞中获得表达,q PCR结果表明在m GP73过表达时mTOR的转录水平随之上升。结论:构建了pc DNA3.1-Flag-m GP73真核表达质粒,m GP73 m RNA过表达时mTOR的m RNA水平也上升,这为进一步研究m GP73在小鼠肝脏肿瘤转移过程中的作用奠定了基础。     

铁蛋白重链亚铁氧化酶活性突变体的构建及鉴定

[目的]构建小鼠铁蛋白重链(ferritin heavy chain,FTH)亚铁氧化酶活性突变基因的真核表达载体。[方法]针对小鼠FTH基因的亚铁氧化酶活性关键位点(K86、E62和H65),设计并合成6条PCR引物。利用重叠延伸PCR技术,获得小鼠FTH基因的突变体m FTH(K86Q、E62K和H65G),简写为m FTH(3M)。然后将m FTH(3M)插入到实验室已有的C端带Flag标签的真核表达载体pc DNA3中,插入位置为多克隆位点区域中限制性内切酶Bam HⅠ和HindⅢ之间,从而得到亚铁氧化酶活性缺失的FTH真核表达载体pc DNA3-m FTH(3M)-Flag。重组质粒经酶切和测序鉴定正确后,用脂质体lipofectamineTM2000将其转染到RAW264.7细胞中,检测细胞中带有Flag标签的m FTH(3M)蛋白的表达。[结果]重组质粒酶切得到预期片段,测序显示所构建的m FTH(3M)真核表达载体序列正确,细胞内检测到带Flag标签的m FTH(3M)蛋白信号的强烈表达。[结论]实验成功构建了小鼠FTH亚铁氧化酶活性位点基因突变的真核表达载体pc DNA3-m FTH(3M)-Flag。     

人血小板生成素cDNA转导促进小鼠生长与胰岛素样生长因子相关

人血小板生成素 ( TPO)基因转导能刺激小鼠生长 .将编码人 TPO的真核表达质粒pc DNA3 h TPO、野生型 pc DNA3 或生理盐水注射小鼠后肢肌肉 ,称量小鼠的体重、器官重 .用半定量 ELISA检测 TPO转导小鼠血清中胰岛素样生长因子 ( IGF) - 、 和血小板源生长因子( PDGF)水平 .结果发现 ,TPO基因转导能促进小鼠的生长 .这种促生长作用与年龄和性别有关 ,但与动物的种系无关 .8～ 1 2周龄雌鼠在基因转导后 1周生长开始加快 .在第 1、2、3和 4周的升高幅度分别为 4.3%、5.3%、4.1 %和 4.38% ( P分别 <0 .0 0 5、0 .0 0 2、0 .0 1和 0 .0 1 ) .小于 4周龄的幼鼠、大于 1 6周的老龄鼠 ,以及雄性鼠在基因转导后体重无明显改变 .半定量 ELISA分析发现 ,体重增加的成年雌性鼠 ,其血清中 IGFs和 PDGF均升高 .基因转导鼠血液中葡萄糖、甘油三酯及碱性磷酸酶水平升高 .结果表明 ,一些生长因子 ,特别是 IGF- ,介导着 TPO对转基因动物的生长刺激作用 .     

婴儿双歧杆菌对小鼠肉瘤S180的抑制作用

本文主要观察婴儿双歧杆菌(Bif. 189—3)对小鼠皮下移植肉瘤180(S180)的抑制作用。结果发现,该菌无论在S180移植前或移植后经皮下注射,均能明显抑制皮下移植S180的增长,并使带瘤鼠存活期显著延长。病理检查见实验组肿瘤坏死明显,肿瘤周围有大量炎症细胞浸润。提示该菌能非特异性增强肿瘤局部的免疫反应。     

人白细胞介素18的基因克隆与表达

人白细胞介素１８（ＩＬ－１８）是新近发现的细胞因子之一．研究表明它参与Ｔ１辅助细胞介导的细胞免疫．利用ＲＴ－ＰＣＲ技术从人外周血细胞扩增得到了ＩＬ－１８的ｃＤＮＡ并测定其核酸序列．利用基因重组技术构建ＩＬ－１８的表达载体,并在大肠杆菌中进行了表达．这为以后进一步研究ＩＬ－１８的功能奠定了基础．     

富硒转白细胞介素2基因双歧杆菌对小鼠移植瘤H22的治疗作用

目的评价富硒和转白细胞介素2（IL2）基因双歧杆菌对小鼠移植瘤H22的抑制效果。方法通过电转导将含IL2质粒导入到长双歧杆菌（Bifidobacterium longum,B.longum）中,将转IL2基因双歧杆菌接种到添加了亚硒酸钠（Na2SeO3）的培养基中,利用微生物的富集及转化作用,形成富硒的含IL2基因质粒的双歧杆菌（Se-B.longum-IL2）。结果利用双歧杆菌的肿瘤厌氧区靶向性,通过荷肝癌H22的小鼠尾静脉注射Se-B.longum-IL2,取得了良好的抑瘤效果与荷肝癌小鼠生存延长效果。结论转IL2基因双歧杆菌和富硒联合后对小鼠肝癌有明显的基因治疗前景。     

Protective immunity against Taenia crassiceps murine cysticercosis induced by DNA vaccination with a Taenia saginata tegument antigen

This study investigated the protective capacity of the recombinant Taenia saginata Tso18 antigen administered as a DNA vaccine in the Taenia crassiceps murine model of cysticercosis. This Tso18 DNA sequence, isolated from a T. saginata oncosphere cDNA library, has homologies with Taenia solium and Echinococcus sp. It was cloned in the pcDNA3.1 plasmid and injected once intramuscularly into mice. Compared to saline-vaccinated control mice, immunization reduced the parasite burden by 57.3-81.4%, while lower levels of non-specific protection were induced in control mice injected with the plasmid pcDNA3.1 (18.8-33.1%) or a plasmid with irrelevant construct, pcDNA3.1/3D15 (33.4-38.8%). Importantly, significant levels of protection were observed between the pcDNA3.1/Tso18 plasmid and pcDNA3.1/3D15 plasmid immunized mice. Mice immunized with pTso18 synthesized low levels of, primarily IgG1 sub-class, antibodies. These antibodies were shown to recognize a 66 kDa antigen fraction of T. crassiceps and T. solium. Splenocytes enriched in both CD4+CD8- and CD4-CD8+ T cells from these vaccinated mice proliferated in vitro when exposed to antigens from both T. solium and T. crassiceps cestodes. Immunolocalization studies revealed the Tso18 antigen in oncospheres of T. saginata and T. solium, in the adult tapeworm and in the tegument of T. solium cysticerci. The protective capacity of this antigen and its extensive distribution in different stages, species and genera of cestodes points to the potential of Tso18 antigen for the possible design of a vaccine against cestodes.     

No influence of tumor growth by intramuscular injection of hepatocyte growth factor plasmid DNA: safety evaluation of therapeutic angiogenesis gene therapy in mice

Recently, a novel therapeutic treatment for ischemic diseases using angiogenic growth factors to augment collateral artery development has been proposed. As intramuscular injection of naked human hepatocyte growth factor (HGF) plasmid DNA induced therapeutic angiogenesis in several animal test subjects, we have started a clinical trial to treat peripheral arterial disease. However, one might assume that over-expression of angiogenic growth factors could enhance tumor growth. To resolve this issue, we examined the over-expression of HGF in tumor bearing mice. Tumors on their backs were prepared with an intradermal inoculation of A431, human epidermoid cancer cells expressing c-Met. These mice were intramuscularly injected with human HGF plasmid or control plasmid into the femoral muscle. Human HGF concentration was increased only in the femoral muscle, but not in blood. Although recombinant HGF stimulated the growth of A431 cells in vitro, temporally and locally HGF elevation in hindlimb had no effect on tumor growth in mice.     

In vivo antitumor effects of murine interferon- γ -inducing factor/interleukin-18 in mice bearing syngeneic Meth A sarcoma malignant ascites

Interferon-γ-inducing factor/interleukin-18 is a novel cytokine that reportedly augments natural killer (NK) activity in human and mouse peripheral blood mononuclear cell cultures in vitro and has recently been designated IL-18. In this study, IL-18 exhibited significant antitumor effects in BALB/c mice challenged intraperitoneally (i.p.) with syngeneic Meth A sarcoma when administered i.p. on days 1, 2 and 3 after challenge. Intravenous (i.v.) administration also induced antitumor effects in the tumor-bearing mice; however, subcutaneous (s.c.) administration did not. When mice were twice pretreated with 1 μg IL-18 3 days and 6 h before tumor challenge, all mice survived whereas control mice died within 3 weeks of challenge. Inhibitory effects on Meth A cell growth in vitro were not observed with either IL-18 or interferon γ. The effects of IL-18 pretreatment were abrogated by abolition of NK activity after mice had been injected with anti-asialo GM1 antibody 48 h before and, 24 h and 72 h after tumor challenge. Mice pretreated with IL-18 and surviving tumor challenge resisted rechallenge with Meth A cells but could not reject Ehrlich ascites carcinoma, and spleen cells from the resistant mice, but not control mice, exhibited cytotoxic activity against Meth A cells in vitro after restimulation with mitomycin C-treated Meth A cells for 5 days. The effector cells in the spleen cell preparations from resistant mice appear to be CD4⁺ cells because cytolytic activity was significantly inhibited after depletion of this subset by monoclonal antibodies and complement. In conclusion, IL-18 exhibits in vivo immunologically (primarily NK) mediated antitumor effects in mice challenged with syngeneic Meth A sarcoma and induces immunological memory and the generation of cytotoxic CD4⁺ cells. Received: 17 September 1996 / Accepted: 8 November 1996     

Oncolytic Adenovirus Expressing IL-23 and p35 Elicits IFN-γ- and TNF-α-Co-Producing T Cell-Mediated Antitumor Immunity

Cytokine immunogene therapy is a promising strategy for cancer treatment. Interleukin (IL)-12 boosts potent antitumor immunity by inducing T helper 1 cell differentiation and stimulating cytotoxic T lymphocyte and natural killer cell cytotoxicity. IL-23 has been proposed to have similar but not overlapping functions with IL-12 in inducing Th1 cell differentiation and antitumor immunity. However, the therapeutic effects of intratumoral co-expression of IL-12 and IL-23 in a cancer model have yet to be investigated. Therefore, we investigated for the first time an effective cancer immunogene therapy of syngeneic tumors via intratumoral inoculation of oncolytic adenovirus co-expressing IL-23 and p35, RdB/IL23/p35. Intratumoral administration of RdB/IL23/p35 elicited strong antitumor effects and increased survival in a murine B16-F10 syngeneic tumor model. The levels of IL-12, IL-23, interferon-γ (IFN-γ), and tumor necrosis factor-α (TNF-α) were elevated in RdB/IL23/p35-treated tumors. Moreover, the proportion of regulatory T cells was markedly decreased in mice treated with RdB/IL23/p35. Consistent with these data, mice injected with RdB/IL23/p35 showed massive infiltration of CD4⁺ and CD8⁺ T cells into the tumor as well as enhanced induction of tumor-specific immunity. Importantly, therapeutic mechanism of antitumor immunity mediated by RdB/IL23/p35 is associated with the generation and recruitment of IFN-γ- and TNF-α-co-producing T cells in tumor microenvironment. These results provide a new insight into therapeutic mechanisms of IL-12 plus IL-23 and provide a potential clinical cancer immunotherapeutic agent for improved antitumor immunity.     

人白细胞介素—18基因的克隆及其在大肠杆菌中的表达

利用反转录ＰＣＲ（ＲＴＰＣＲ）法从人肝组织中分离人白介素１８（ｈＩＬ１８）基因。克隆到Ｔｖｅｃｔｏｒｅａｓｙ载体中,经酶切初步鉴定后进行ＤＮＡ序列分析,结果表明与文献报道完全一致。以ｐＢＶ２２０为表达载体,在大肠杆菌中高效表达了ｈＩＬ１８基因,重组蛋白占菌体总蛋白的２７．２％。为进一步研究其生物活性奠定了重要基础。     

质粒DNA增强大鼠缺血骨骼肌肌浆网钙转运功能

在大鼠肢体缺轿模型上观察质粒ｐｃＤＮＡ３对缺血骨骼肌肌浆网（ＳＲ）Ｃａ＾２＋转运的影响。结果显示,骨骼肌缺血时ＳＲＣａ＾２＋转运（Ｃａ＾２＋摄入与释放）较非缺血肌肉增强,而质粒ｐｃＤＮＡ３与ＳＲ上ＤＮＡ结合蛋白结合之后,可进一步增强缺备骨骼肌ＳＲＣａ＾２＋摄入（Ｐ＜０．０１）及释放速率（Ｐ＜０．０５）。提示质粒ＤＮＡ对正常及缺血大鼠骨骼肌的ＳＲＣａ＾２＋转运能力均有影响,其临床病理生理意义值得进一