Construction of eukaryotic expression vector of fusion gene CD80-IgG and its expression in Chinese hamster ovary cells

AIM: To construct the eukaryotic expression vector CD80-IgG by fusing the cDNA encoding extracellular portion of murine CD80 to the 5'-terminus of cDNA encoding Fc fragment of murine immunoglobulin G1 and to express the fusion protein in Chinese hamster ovary (CHO) cells. METHODS: The two cDNAs was amplified by PCR respectively from plasmid pcDNA/B7 containing the full-length cDNA of murine CD80 from murine spleen cells, and cloned to the eukaryotic expression vector pcDNA3.0 by directional cloning. The resultant recombinant plasmid pcDNA/CD80-IgG was transfected into CHO cells with liposome transfection reagent. The stably expressing cells were obtained by G418 screening. Western blot, Dot ELISA, and flow cytometry were used to detect the expression of the fusion protein and its immunological activity. RESULTS: DNA sequencing verified the correction of the construction of recombinant plasmid pcDNA/CD80-IgG. The expressed fusion protein was detected in the supernatant of transfected CHO cells and the molecular weight of the protein was similar to what we expected. Its immunological activity was also established. CONCLUSION: The recombinant plasmid pcDNA/CD80-IgG was successfully constructed and it expressed the fusion protein CD80-IgG.     

狗牙根种质对冷害的生理反应

对采自佛山市的3个狗牙根材料(狗牙根1号,3号和5号)及杂交狗牙根品种天堂419的抗寒性进行了比较研究,发现田间种植的狗牙根材料在受到2004年12月的寒潮袭击后,相对电导率升高,其中天堂419的升高幅度最大,而狗牙根1号和5号的升高幅度较小;天堂419的光合速率降低最多,而狗牙根5号的光合速率仅轻微降低;叶片的可溶性糖含量均提高,狗牙根5号的根系可溶性糖含量升高,但其他材料的可溶性糖含量降低。在韶光地区霜冻条件下的试验结果表明,狗牙根5号的草坪质量评分最高,绿期最长,表明狗牙根5号的抗寒性较强。     

Study on Synergistic Effect of VA5 Immunopotentiator on Pigeon NDV Inactivated Vaccine

This study was designed to evaluate the synergistic effect of VA5 immunopotentiator on pigeon ND4416 inactivated vaccine.160 healthy pigeons were randomly divided into four groups,including ND4416 strain inactivated vaccine group (NDV group),ND4416 strain inactivated vaccine and immunopotentiator (VA5) mixed group (NDV+VA5 group),La Sota inactivated vaccine group (La Sota group) and normal saline as the control group (C group),to assess their vaccine efficacy against virulent pigeon NDV by serological analysis and animal testing.Pigeons sera were collected at different time points after immunization and measured the HI antibody titer of each group.The results showed that VA5 immunopotentiator significantly improved the serum antibody level of pigeons immunized with pigeon Newcastle disease inactivated vaccine (P<0.05).In addition,comparative test of spleen lymphocyte transformation was conducted at various time points after immunization.The results indicated that VA5 effectively stimulated the lymphocyte transformation of immune pigeon.Pigeons in each groups were challenged with ND4416 strain at the 30th,90th and 180th d after immunization.The results presented that the NDV+VA5 group had 100% protection rate and higher than La Sota group.The duration of immunization test showed that the antibody titer of NDV+VA5 group reached peak 11.20log₂ at the 21st d,remained 7.50log₂ at 180th d,and the protection rate remained 100% at 180th d.It indicated that VA5 immunopotentiator sustained the immune duration of pigeon NDV vaccine up to 180 d.Moreover,the in vitro detoxification test results suggested that VA5 immunopotentiator reduced the in vitro detoxification cycle of pigeons after challenge.Overall,this study suggested VA5 immunopotentiator could significantly improve the immune efficacy of pigeon Newcastle disease inactivated vaccine,which provided a basis for further enhancing the efficacy of Newcastle disease vaccine,as well increased experimental data for the application of immunopotentiators.     

“站场调车管理系统“监控中心管理程序的设计

监控中心管理程序主要包括五大模块:权限管理模块、用户管理模块、调机作业单模块、站场基本数据管理模块、站场信息及查询模块.它能够将系统的“监控处理程序“所产生的站场各种资料,在监控中心的计算机屏幕上真实的反映站场当前的各种信息,以及再现站场的历史信息.     

Hesperetin attenuates hypoxia/reoxygenation-induced apoptosis in H9c2 cells via inhibition of calcium overload

AIM: To investigate the effect of hesperetin on hypoxia/reoxygenation (H/R)-induced apoptosis in the H9c2 cells and to clarify the underlying mechanism. METHODS: The H/R model was established and the H9c2 cells were pretreated with hesperetin for 4 h. The cell viability and cell damage were measured by CCK-8 assay and lactate dehydrogenase (LDH) detection. The apoptosis was analyzed by Hoechst 33258 staining and flow cytometry. The intracellular calcium fluorescence intensity was measured by fluorescence microscopy and flow cytometry. The calcium-ATPase activity and the level of adenosine triphosphate (ATP) were measured by ELISA. The mitochondrial membrane potential was measured by JC-1 staining. The protein expression levels of Bcl-2, Bax and cytochrome C (Cyt-C) were determined by Western blot. RESULTS: Hesperetin reduced the apoptosis of the H9c2 cells induced by H/R, decreased intracellular Ca²⁺ fluorescence intensity, elevated Ca²⁺-ATPase activity, inhibited the mitochondrial membrane potential depolarization and increased the level of ATP (P<0.05). In addition, hesperetin significantly reduced the release of Cyt-C protein from mitochondria to cytoplasma and increased the Bcl-2/Bax ratio (P<0.05). After using the calcium ion inhibitor nimodipine, the percentage of the cells with mitochondrial membrane depolarization was decreased, the ATP level was increased and the protein expression of mitochondrion-related apoptosis molecules were decreased (P<0.05). CONCLUSION: Hesperetin reduces the apoptosis of the H9c2 cells induced by H/R, which may be related to inhibition of calcium overload and improvement of mitochondrial function.     

桑树花青素合成酶(ANS)基因的克隆及在2种果色桑树中的表达特征

花青素合成酶(anthocyanidin synthase,ANS)是植物花青素生物合成途径末端的关键酶,催化无色花色素到有色花色素的转变。从广东桑品种粤椹大10中克隆得到一条ANS序列,其ORF为1 077 bp,编码358个氨基酸,预测蛋白质分子质量为41 kD,等电点为5.62,具有2-酮戊二酸双加氧酶的保守结构域。多重序列比对表明物种间的ANS序列高度保守,系统进化树中桑树ANS与芍药科、芸香科、葡萄科等植物的同源序列聚在一个类群上。RT-PCR检测桑树ANS在结紫色果的粤椹大10的幼叶和桑椹中特异性表达,并且随着果色加深其表达水平呈上升趋势,而在结白色果的桑品种珍珠白的幼叶和桑椹中检测不到ANS的表达,暗示桑树ANS在桑椹的颜色形成中起到重要作用。     

兽药产品批准文号申报核发及管理有关问题探讨

兽药产品批准文号管理是我国控制兽药市场准入的前置性管理模式,本文就目前兽药产品批准文号申报、核发及管理中存在的一些问题进行了分析,对企业申报兽药产品批准文号时的注意事项进行了阐述。     

三江源高寒退化草原土壤有机质含量的光谱模拟估算

以三江源玛多县不同退化程度高寒草原土壤为研究对象,采集0～30 cm土层的90个土壤样品,测定土壤样品的光谱反射率和有机质（SOM）含量,分析不同光谱数据转换方式与土壤SOM含量的相关性,据此挑选P<0.001水平的显著波段作为特征波段,并与土壤SOM含量建立多元逐步回归（MLSR）、支持向量机（SVM）、决策树（DT）、随机森林（RF）模型。结果表明：1）高寒草原土壤SOM含量属中等变异,且与土壤原始反射率呈负相关,与倒数对数呈正相关;2）光谱数据的数学转换扩大了光谱的吸收特征,log(1/R)、R’、[log(1/R)]’与土壤SOM含量相关系数绝对值的最大值比R分别提高了0.099、0.156、0.160;3)RF反演模型精度高于其他反演模型,Log(1/R)-RF模型的预测效果较好,其建模组和检验组的决定系数（R2）、均方根误差（RMSE）分别为0.949 1、0.252 69和0.717 23、0.496 9,可以准确估算高寒草原土壤SOM的含量。     

“贵长”猕猴桃花粉直感效应研究

为了探讨不同授粉树对“贵长”猕猴桃着果率及果实品质的影响,进而为科学选配授粉树提供依据,从贵州省水城县及修文县选择18个不同品种(株系)授粉树,比较花粉性状并保存花粉,在“贵长”猕猴桃雌花盛花期时进行人工授粉,比较不同花粉授粉下的果实品质差异。结果表明,“贵长”猕猴桃在单果质量、种子数、种子质量、种子大小、果形、可溶性糖含量、可滴定酸含量、可溶性固形物含量、维生素C含量、可溶性蛋白含量以及干物质含量方面均表现出明显的花粉直感效应,而果皮茸毛未表现出花粉直感效应。猕猴桃单果质量受种子数、种子大小、种子质量及其他因素共同影响。多数情况下,高倍体作为父本表现出高着果率。在供试雄株中,“GX1”和“GX12”给“贵长”猕猴桃授粉的综合表现较好,适合作为“贵长”猕猴桃的配套授粉树。     

猪伪狂犬病病毒双基因缺失突变株(HB-98株)安全性、稳定性和免疫原性测定

对以伪狂犬病病毒鄂A株为亲本毒株构建的TK和gG双基因缺失突变株（PrV HB-98株）的增殖能力、安全性、毒力稳定性和免疫原性进行了测定。结果表明，PrV HB-98株在BHK-21细胞上的增殖滴度为10^7.0 TCID50／0．1mL以上，与亲本毒株相当，但高于Bartha株；与PrV鄂A株相比，病毒量为10^7.0TCID50的PrV HB-98株不引起BALB／c小鼠的死亡，毒力也低于Bartha株；将PrV HB-98株在PK-15细胞连续培养25代和在猪体内上连续继代5次，各代次突变株TK基因和LacZ基因能被稳定扩增，未出现毒力回复现象．表明该毒株具有良好的遗传稳定性；以10^5.0、10^6.0、10^7.0TCID50等3个不同剂量的PrV HB-98株接种于妊娠50～60d母猪和1日龄仔猪，母猪均能正常产仔．仔猪也未出现任何临床症状，证明该毒株有较好的安全性。另外，以10^5.0TCID50的PrV HB-98株接种于妊娠50～60d母猪和1日龄仔猪，分别于接种后28d和20d，用10^7.0TCID50 PrV鄂A强毒进行攻击．结果免疫猪都能抵抗强毒的攻击．获得保护，表明该毒株具有很强的免疫原性。综合上述结果表明，PrV HB-98株可以作为候选毒株．用于伪狂犬病基因工程疫苗的研制。