Construction and anti-tumor effects of recombinant fowlpox virus expressing Newcastle disease virus hemagglutinin-neuramidinase gene

Hemagglutinin-neuramidinase (HN), a Newcastle disease virus-derived protein, not only mediates receptor recognition but also possesses neuraminidase (NA) activity, the ability to cleave a component of those receptors, N-acetylneuraminic acid (NAcneu, sialic acid). It is known that this protein in mammalian species, including human beings, has interesting anti-neoplastic as well as immune stimulating properties. To explore the use of the HN gene in cancer gene therapy, we constructed a recombi-nant fowlpox virus expressing the HN protein (vFV-HN) and compared the anti-tumor activity of the recombinant virus with that of wild-type fowlpox virus (FPV) in vivo and in vitro. Here we found that although B16 cells were somewhat resistant to the basal cytotoxic effect of wild-type fowlpox virus, infection with vFV-HN caused a pronounced cytotoxic effect and, the survival of tumor-bearing mice immunized with vFV-HN was significantly increased compared with the survival of mice immunized with the FPV alone. Furthermore, the immunization of mice with vFV-HN elicited a B16 tumor-specific cytotoxic T lymphocyte (CTL) response and clonal expansion of both CD4 and CD8 T cell populations in vivo. In addition, T cells from lymph nodes of mice vaccinated with vFV-HN secreted high levels of the Th1 cytokine IL-2 and IFN-γ, indicating that the regression of tumor cells is related to a Th1-type dominant immune response. These results demonstrate that vaccination with vFV-HN may be a potential strategy for cancer gene therapy.     

Research advances in gene therapy for Parkinson's disease

During the long period of time when people have been seeking for an effective therapeutic method for PD, the gene therapy has shown greater and greater advantages over other methods. It can be performed mainly in two ways: ex vivo and in vivo. With the former, TH gene as well as some neurotrophic factor genes (such as GDNF, BNDF genes) can be invited to some cell lines or primary cells thus forming engineered cells and then implanting them into brain. While with the latter, viral vectors including HSV-1, Ad, AAV that can be utilized to construct recombinant viruses, or non-virus vectors can be used to delived DNA into brain directly. The present review summarizes the recent research advances in the gene therapy for PD, and it is reasonable for us to predict a notable progress in prevention and treatment for PD in the next decade.     

基因表达系列分析在肿瘤研究中的应用

基因表达系列分析(serial analysis of gene expression, SAGE)是一种快速分析基因表达信息的技术.它不但能快速、详细地分析成千上万个基因,还能发现新基因,因此是基因表达定性和定量研究的一种新的有效手段.近年来此技术广泛应用于肿瘤的研究,了解肿瘤发病机制,识别诊断和治疗肿瘤的新基因.可以预测SAGE在肿瘤研究和诊断过程中的应用将会对肿瘤的认识和治疗产生深远的影响.     

Specific anti-tumor effect induced by attenuated Salmonella typhimurium vaccine expressing extracellular region of vascular endothelial growth factor receptor 2

The purposes of this research were to study the stable expression of exogenous gene encoding therapeutic protein in attenuated Salmonella typhimurium, observe the metabolism of oral gene vaccine carried by attenuated Salmonella typhimurium in BALB/c mouse, and investigate the feasibility of prevention and treatment of tumors by the recombinant bacteria. Recombinant plasmid pcDNA3.1 VEGFR2(n1-7) was transformed into competent attenuated Salmonella typhimurium SL3261 to develop oral DNA vaccine SL3261-pcDNA3.1 VEGFR2(n1-7). To observe whether the exogenous gene can be expressed in the recombinant bacteria, PCR was performed to amplify the CMV promoter of the eukaryotic expression vector as the proof of stable expression of exogenous protein; transmission elec- tron microscopy (TEM) was applied to observe the morphology of the recombinant bacteria to confirm that the exogenous gene has no impact on the growth of the bacteria, and then BALB/c mice were immunized with the gene vaccine. After inoculation of the gene vaccine, the recombinant bacteria SL3261 could be detected in the tissues such as small intestine, colon, liver and spleen. And then, mice in each group were challenged with tumor cells. The results of animal experiment showed that tumor growth of the mice in experimental group was inhibited and survival time of immunized mice was prolonged compared with control groups. A higher lymphocyte infiltration in tumors from animals treated with DNA vaccine was observed. Immunohistochemical analysis of tumor samples revealed an en- hanced accumulation of CD8 cytotoxic T lymphocytes, as well as an increase in CD4 cells in the tumors of animals treated with the oral gene vaccine compared to tumors from control group mice. Ultrastructure of the tumor tissue showed that tumor cells in the samples of the immunized mice were well-differentiated. Our research confirmed that the exogenous gene can be stably expressed in the attenuated Salmonella typhimurium and has no impact on the growth of the recombinant bacteria; the exogenous gene can de delivered to the host by attenuated Salmonella typhimurium to produce anti-tumor effect with no obvious cytotoxity to the host. In this study, it is established that attenuated Salmonella typhimurium could be used as a vector for oral gene vaccine, and our study provided a theoretical basis for the body distribution and the metabolism of the recombinant bacteria. This strategy may provide a simple, safe and effective way for the prevention and treatment of tumors.     

中国荷斯坦牛催乳素基因A8398G多态性与产奶性状相关性分析

采用PCR-RFLP技术对中国荷斯坦牛PRL基因第4外显子A8398G位点进行单核苷酸多态性分析。结果表明PRL基因经RsaⅠ酶切后产生2种等位基因和3种基因型。等位基因A和G的频率分别为0.257和0.743。基因型AA、AG和GG的频率分别为0.010,0.497及0.493。测序分析表明AA型和GG型相比在PRL基因第8398位碱基处发生A→G突变,该突变未导致氨基酸的改变。χ2适合性检验表明,PRL基因的RsaⅠ酶切位点处于Hardy-Weinberg不平衡状态(P<0.05)。在第Ⅱ泌乳期,最小二乘分析表明:基因型GG所对应的乳蛋白率最小二乘均值显著高于基因型AG所对应的最小二乘均值(P<0.05);基因型AG所对应的产奶量最小二乘均值显著高于基因型GG所对应的最小二乘均值(P<0.01);基因型GG所对应的线性评分最小二乘均值显著高于基因型AG所对应的最小二乘均值(P<0.05)。     

靶向hTERT的shRNA腺病毒载体的构建及其对MCF-7细胞hTERT表达的影响

设计针对人端粒酶逆转录酶催化亚基(hTERT)的干扰靶序列GGAACACCAAGAAGTTCATCT,构建siRNA表达质粒PgenesilhTERT;随后将shRNA表达框克隆到入门载体pENTRTM1A,构建重组质粒pENTR/U6-hTERT-polyA;使用同源重组方式在体外将该表达框重组到腺病毒载体pAd/PL-DEST,得到重组腺病毒质粒pAd/U6 –TERT-polyA;线性化的重组腺病毒质粒在HEK 293细胞内包装为具有感染能力的病毒颗粒rAd-hTERT.同时构建不针对任何基因的shRNA阴性对照rAd-HK,空病毒对照rAd-blank和只含EGFP的rAd -EGFP.四种病毒经酶切、电泳分析表明插入序列正确,腺病毒载体构建成功.Western blotting测定转染后各组hTERT蛋白的表达情况,证实转染rAd-hTERT 48 h后,hTERT的表达明显被抑制.实验表明基于Gateway技术的腺病毒介导的shRNA载体构建成功,rAd-hTERT可有效抑制乳腺癌MCF-7细胞hTERT基因的表达.本研究为针对端粒酶的基因治疗奠定了实验基础.     

Re-evaluation of the phylogenetic position of the genus Dexiotrichides (Protozoa, Ciliophora, Scuticociliatida) inferred from stomatogenetic and molecular information for Dexiotrichides pangi

The divisional process and systematic position of the marine scuticociliate Dexiotrichides pangi are studied. Based on both stomatogenetic data and 18S rDNA gene sequences, the phylogeny and morphogenetic characteristics of this taxon, and of other related genera, are analyzed and discussed. Both the divisionary events and the molecular biological data indicate that this speciesgenus, together with certain other genera in the Dexiotricha-complex, occupies an intermediate position between the tetrahymenids and the “typical” scuticociliate, which suggests that the Dexiotricha-like taxa should be excluded from the “true” scuticociliates. As a further contribution, the process of stomatogenesis in D. pangi can be summarized as follows: (1) The oral primordia in the opisthe are formed only by the proliferation of basal bodies in the scutica field, which subsequently develop into three membranelles, while the new paroral membrane seems to be generated by the sub-anterior portion of somatic kinety 1 (the 1st postoral intercalary kinety). The latter character exhibits a mode similar to Tetrahymena. (2) In the proter the parental membranelles are retained and remain unchanged throughout the entire division process; only the old paroral membrane is disassembled and differentiated into the anlage, which then gives rise to the new paroral membrane and the scutica of the proter. The 18S rRNA gene sequence reported here is the first one for a ciliate in the Dexiotricha-complex.     

矢竹地下茎转录组测序及节间生长相关基因表达分析

【目的】揭示竹子地下茎生长发育的分子机制。【方法】利用高通量转录组测序对矢竹含不同发育阶段节的地下茎转录组图谱进行了分析研究,并利用qRT-PCR对节间生长相关基因进行表达模式分析。【结果】共获得了11 976 条平均长度为1 008 bp的单基因簇(unigenes)。NCBI注释显示,63.8%的单基因簇具有注释结果,其中16 254 条unigenes被注释到137 个KEGG(京都基因与基因组百科全书)代谢通路中。MapMan分析共获得了1 864 个转录因子及603 个激素代谢及信号转导相关单基因簇。在功能基因方面,共获得564 个与细胞壁构建相关的代谢基因,其中92 个与木质素合成相关。此外,对9 个与赤霉素、细胞壁合成及细胞骨架组织相关的基因,通过实时荧光定量PCR,分析了它们在节间不同发育阶段的表达模式。结果显示:细胞壁、细胞骨架下游功能相关基因在矢竹地下茎节间快速生长阶段表达最高; 而赤霉素合成及信号传导相关基因则在节间伸长起始阶段最高。【结论】矢竹地下茎生长发育受到从各类激素、转录因子到其下游功能基因等组成的复杂分子网络的协同调控。     

Mining rice new germplasm containing S5n gene by functional molecular marker and sequencing

Inter-subspecific hybrids between indica and japonica varieties yield strong biological heterosis, but it is difficult to utilize the hybrids directly in commercial production due to sterility of F1. A special gene S5^n may overcome the hybrids sterility, which is caused by the interaction between S5 loci. Recently, S5^n had been cloned, and it was revealed containing a large DNA deletion sequence that made the gene nonfunctional, compared to S5^i or S5^j. We designed a pair of primers flanking the deletion sequence of Ssn, and then applied to distinguish the varieties with S5^n or non-S5^n, convincing result suggested that the primers could be served as functional molecular marker to efficiently identify the new germplasm with S5^n, Using the functional marker, we surveyed 197 varieties from China National Micro-core Rice Collection, and found ten of which represented S5^n including following varieties： Haobuka, Sanbangqishiluo, Mubanggu, Xiaohonggu, Mowanggu＇neiza, Laozaogu, Fanhaopi, Feie＇nu02, Baoxie-7B, Teqingxuanhui. Among them, two varieties Sanbangqishiluo and Laozaogu was previously reported to contain S5^n gene. Further sequence analysis on the DNA sequence covering both sides of deletion in S5^n of the 10 varieties confirmed that the detected sequences in above varieties was identical with those of varieties containing S5^n, such as 02428 and Linglun. These results suggested that the gene in S5 locus of the ten varieties was also nonfunctional and it proved the presence of S5^n gene.     

生物工程与现代农业

介绍了世界农业面临的形势与任务,论述了生物工程技术在农业领域应用研究进展,并对我国未来农业发展进行了展望,提出了建议。