马白细胞介素18成熟蛋白(mEIL-18)基因在大肠埃希氏菌中的高效表达与纯化

用RT—PCR从经ConA刺激的马外周血单个核细胞（PBMC）中扩增出马白细胞介素18（Equine interleukin-18，EIL-18）前体蛋白（precursor EIL-18，pEIL-18）基因的cDNA，然后克隆至载体pCR2．1-TOPO中，鉴定并命名为pCR2．1-pEIL-18。自pCR2．1-pEIL-18中扩增马白细胞介素18成熟蛋白（mature EIL-18，mEIL-18）基因，并将其亚克隆至原核表达载体pET-28a（＋）中。将筛选出的阳性克隆进行测序、诱导表达并纯化其表达产物。结果表明，mEIL-18基因全长474bp，含1个开放阅读框，编码157个氨基酸的成熟蛋白；表达产物以可溶性和包涵体两种形式存在，经SDS-PAGE和Western—blot分析，重组蛋白相对分子量约为20ku，且具有免疫生物学活性。mEIL-18基因在大肠杆菌中高效表达并在非变性和变性条件下采用Ni^＋亲和层析纯化方法获得了高纯度的重组mEIL-18，为探究马白细胞介素18的生物学活性及其应用奠定了基础。     

猪传染性胃肠炎病毒TaqMan荧光定量RT-PCR检测方法的建立

根据猪传染性胃肠炎病毒和猪肌动蛋白的基因序列设计合成了引物和探针,通过对荧光定量RT-PCR反应条件的优化,建立了TaqMan荧光定量RT-PCR检测TGEV的方法。同时对37份现地病料进行检测并与常规RT-PCR方法、TGEV抗原快速检测试剂盒比较。结果,该方法的检测敏感性达到15.3拷贝/μL,且具有很好的特异性和重复性,而常规RT-PCR方法只能检测到1.53×103拷贝/μL。对37份现地病料的检测结果也表明该法(检出17份)比常规RT-PCR方法(检出12份)和TGEV抗原快速检测试剂盒(免疫层析法,检出10份)的敏感性高。     

禽传染性支气管炎DNA疫苗在鸡体内的分布和安全性

采用本实验室构建的3种禽传染性支气管炎病毒基因的真核表达质粒pIBVS1、pIBVM、pIBVN,按各50 mg/L配制成质量浓度为150 mg/L的DNA疫苗,经腿部肌肉分点注射免疫1周龄SPF雏鸡。分别在免疫后24 h,73、0及60d,采集试验鸡的血液、心、肝、脾、肺、肾、胸腺、性腺（卵巢/睾丸）及注射部位肌肉进行组织总DNA抽提,以组织总DNA为模板进行PCR扩增。另外,在对照组DNA模板中加入不同拷贝数的质粒,确定PCR反应的灵敏性。以纯化后的组织总DNA为模板,PCR法检测质粒DNA整合到鸡细胞染色体基因组上的可能性,评价疫苗的安全性。结果表明,该疫苗在24 h内迅速分布于全身,并能在血液及所有检测的组织器官内持续分布60 d;纯化后的组织基因组DNA经PCR扩增均呈阴性,未发现整合现象,证实该DNA疫苗的安全性好。     

地下长廊蜜蜂越冬技术的研究

依据蜜蜂室内外越冬原理和蜜蜂生物学特性,按养蜂生产的需要,研究设计了一种蜜蜂越冬的地下长廊,两侧放置1～3层蜂箱,中间为通道,上部封闭,留有进出气孔调节温度,人可以进入长廊管理越冬蜂群.挖建长廊的成本低,越冬期温度稳定,越冬蜂群势削弱率降低20.1%～24.3%,饲料消耗量降低10.5%～16.2%.     

大脑皮质神经元培养中胰蛋白酶消化分离技术的探讨

为探讨大脑皮质神经元培养中胰蛋白酶消化分离法的最佳条件, 取新生大鼠大脑皮质组织，在不同的胰蛋白酶消化条件下分离培养神经元，通过细胞形态观察以及染色计数比较不同消化条件对神经元活性的影响。结果表明，0.25％胰蛋白酶，37℃，5 min消化分离的神经元活性较好，细胞产率也较高。在新生大鼠大脑皮质神经元培养时，采用0.25％胰蛋白酶，37℃，5 min可消化分离到单层、生长活性较好的神经元。     

A群猪轮状病毒JS株vp7基因序列分析

根据已发表的猪轮状病毒OSU毒株vp7基因核苷酸序列ORF两端保守区序列,设计一对特异引物,以猪轮状病毒JS毒株反转录cDNA为模板,通过PCR方法扩增出长约1000 bp目的片段。将其进行T-A克隆、序列测定和分析。结果表明,vp7基因全长1062 bp,含有一个981 bp的开放阅读框,编码326个氨基酸。与已知的15个毒株vp7全长基因的核苷酸及推导的氨基酸序列比较,同源性分别为74.5%～78.5%和75.2%～83.1%,核苷酸系统发育进化树结果表明,JS毒株与轮状病毒G9型参考毒株ICB2185、O-1亲缘关系较近,分为一个群,表明JS毒株血清型为G9型。目前,我国尚未见猪及其它动物轮状病毒G9型流行株的报道。     

鸡胚中沉积的尿酸盐对血凝试验的影响

在新城疫La Sota半成品检验过程中,发现接种后培养120 h的活胚,经过-20℃低温速冻1 h,有的鸡胚尿囊液中有尿酸盐产生,且出现的几率比较大,而经过2~8℃低温冷冻24 h的鸡胚,产生白色尿酸盐的几率相对小一些。凡是有尿酸盐产生的鸡胚,均没有血凝现象产生。但用含有尿酸盐的尿囊液接种鸡胚,继续增殖,会有病毒大量复制,试验说明有新城疫病毒存在的尿囊液中,若有尿酸盐沉淀,则血凝现象会被遮盖。     

遮光对青榨械光合速率及叶绿素荧光参数的影响

研究了遮光15％、35％、50％和全光照处理对青榨槭新梢生长、叶片色素含量、净光合速率以及叶绿素荧光参数的影响。结果表明，随着遮光度增加，叶片色素含量增加，叶绿素a／b减小；叶片净光合速率（Pn）、初始荧光（Fo）、原初光能转化效率（Fv／Fm）和光化学猝灭系数（qP）增加，但非光化学猝灭系数（qN）降低。全光照和遮光15％处理条件下，Pn日变化呈双峰曲线，而35％和50％处理条件下呈单峰曲线；遮光35％和50％处理条件下Fv／Fm日变化幅度较小，15％和全光照处理条件下Fv／Fm日变化呈先上升后下降再上升趋势。50％遮光处理青榨槭幼苗Pn最高，新梢年生长量最大，可在育苗生产中应用。     

Distribution and identification of immunocytes in humanized SCID mouse model

AIM: Humanized-NOD/SCID(hu-NOD/SCID) mouse model was established and the level of immune reconstitution was assessed in this model. METHODS: Mononuclear cells (MNC) and CD34⁺ cells were isolated or sorted from cord blood(CB). Human CD45, CD19, CD3 markers on cells from NOD/SCID murine peripheral blood(PB), bone marrow(BM), thymus were detected by FCM from 4 to 10 weeks after hematopoietic stem cell transplantation. After 10 weeks, the gene expressions of the human β₂M and RAG2 were detected by RT-PCR in PB or bone marrow of mice model. RESULTS: Human CD45, CD19, CD3 cells populations in PB and BM were found by flow cytometry in mice model transplanted with CD34+ cells or CB MNC from 4 to 10 weeks. The highest positivity of human lymphocytes was at 8 week after transplantation. The levels of human cell engraftment in mice transplanted with CD34⁺ cells were higher than those in mice transplanted with CB MNC. The mRNA of human β₂M and RAG2 were found by RT-PCR in BM.CONCLUSION: The higher level of human lymphocyte engraftment is established in NOD/SCID mouse model transplanted with CD34⁺ compared with CB MNC. The maturation of T lymphocytes could be happened in bone marrow of mice model.     

Nucleostemin gene expression in human breast tumor tissues and its relation with clinical pathology

AIM: To study the expression of nucleostemin (NS) gene in human breast tumor tissues and the relations of NS gene expression level with histological grades,histological types and TNM stages of the tumor.METHODS: Total RNA was isolated from human breast tumor tissue.The methods of electrophoresis and RT-PCR were used in measuring NS gene expression level,and the relations of NS gene expression level with histological grades,histological types and TNM stages of the tumor were analyzed.RESULTS: The results indicated that there was no NS gene expression detected in normal breast tissues,and NS gene expression in malignant breast tumor tissues (P<0.01) was higher than that in the benign breast tumor tissues.The higher histological grades of the breast cancer showed the stronger NS gene expression (P<0.01),the higher TNM stages of the breast cancer showed the stronger NS gene expression (P<0.01),and the level of NS gene expression had not correlation with the histological types (P>0.05).CONCLUSION: It is suggested that there is no relation of NS gene expression level with histological types of the breast cancer,but there is a marked correlation of NS gene expression level with the histological grades and TNM stages.