LHRH-PE40的免疫原性及其抗体对细胞毒作用的影响

目的观察连续静脉注射重组毒素LHRH-PE40所引起的家兔抗LHRH-PE40抗体产生的规律以及该抗体对细胞毒作用的影响。方法将家兔分为3组,第1组隔日连续静脉注射LHRHPE40,第2组用弗氏佐剂乳化的LHRHPE40皮下注射为阳性对照组,第3组静脉注射人血白蛋白为阴性对照组。用ELISA检测血清中的抗体水平,XTT检测LHRH-PE40与血清中和后对Hela细胞的细胞毒作用。结果抗LHRHPE40抗体水平随静脉注射时间的延长而升高,8d可以检测到抗LHRH-PE40抗体,在24d基本达到最高水平。血清中和后LHRHPE40对Hela细胞的半数抑制量较阴性血清提高1～2倍,而阳性血清较阴性血清提高14倍。结论连续静脉注射LHRH-PE40产生较低水平的抗体,这种抗体未造成LHRHPE40对Hela细胞半数抑制量产生严重影响,这为临床连续使用LHRH-PE40提供了依据。     

牛心肌肌钙蛋白T的提取、纯化及其单克隆抗体的制备

目的提取、纯化牛心肌肌钙蛋白T(Cardiac troponin T,cTnT),并制备其单克隆抗体。方法采用组织匀浆、蛋白层析技术提取、纯化牛cTnT,紫外分光光度法检测其浓度;SDS-PAGE检测其纯度;间接ELISA法检测其反应特性。将纯化的牛cTnT免疫小鼠,进行细胞融合,间接ELSIA法筛选阳性杂交瘤细胞株,采用小鼠体内法制备腹水。结果纯化的两批牛cTnT浓度分别为0.246和0.336 mg/ml;相对分子质量约为37 000,纯度分别为86.02%和93.77%;纯化的cTnT蛋白可与鼠抗牛cT-nT单克隆抗体结合。共获得5株分泌抗牛cTnT单克隆抗体的杂交瘤细胞株,其中,阳性杂交瘤细胞株3B10腹水抗体效价达1∶10 240。结论已成功提取、纯化了牛cTnT,并建立了稳定分泌抗牛cTnT单抗的杂交瘤细胞株,为cTnT检测试剂盒的国产化奠定了基础。     

狂犬病病毒抗体竞争ELISA检测方法的建立及初步应用

目的建立狂犬病病毒抗体竞争ELISA检测方法,用于不同株狂犬病疫苗免疫后抗体水平的检测。方法用市售的不同狂犬病疫苗株抗原免疫NIH小鼠,制备免疫血清,用不同的疫苗株抗原包被酶标板,分别测定小鼠血清抗体效价,并通过不同株抗原抗体的交叉中和实验,分析抗原的差异性;在此基础上,将不同毒株抗原按不同比例混合包被酶标板,分别测定阳性血清样本,并与快速免疫荧光抑制试验(RFFIT)检测结果比较,确定包被抗原模式,建立狂犬病病毒抗体竞争ELISA检测方法,绘制标准曲线,确定最佳定量范围及灵敏度,确定Cut-off值;并对其特异性、精密性、准确性及稳定性进行验证;用建立的方法与其他市售狂犬病病毒抗体检测试剂分别检测血清样品,分析检测结果的一致性及相关性。结果狂犬病病毒抗原AG∶CTN-1=4∶1为最适包被抗原模式,试剂经优化后,检测抗体效价范围在535.00～33.44 mIU/ml之间,具有良好的线性关系(r>0.99),最低检出限为8.36 mIU/ml,Cut-off值为0.735 mIU。该方法检测人血清白蛋白、破伤风阳性血清、乙肝表面抗原阳性血清、白喉阳性质控血清均未发生反应;试验内变异系数在6.68%～7.84%之间;回收率在97.25%～104.50%之间;试剂置37℃3 d,与4℃保存试剂测定结果差异无统计学意义(P>0.05)。该方法检测血清样品与市售狂犬病病毒抗体检测试剂比较,均具有良好的一致性;与RFFIT测定结果比较,二者差异无统计学意义(P>0.05),回归方程为Y=-0.475+3.246 X,相关系数为0.801。结论已建立了狂犬病病毒抗体竞争ELISA检测方法,可用于大规模狂犬病病毒抗体的筛查。     

A Novel Approach to Revealing Positive and Negative Co-Regulated Genes

As explored by biologists, there is a real and emerging need to identify co-regulated gene clusters, which include both positive and negative regulated gene clusters. However, the existing pattern-based and tendency-based clustering approaches are only designed for finding positive regulated gene clusters. In this paper, a new subspace clustering model called g-Cluster is proposed for gene expression data. The proposed model has the following advantages： 1） find both positive and negative co-regulated genes in a shot, 2） get away from the restriction of magnitude transformation relationship among co-regulated genes, and 3） guarantee quality of clusters and significance of regulations using a novel similarity measurement gCode and a user-specified regulation threshold δ, respectively. No previous work measures up to the task which has been set. Moreover, MDL technique is introduced to avoid insignificant g-Clusters generated. A tree structure, namely GS-tree, is also designed, and two algorithms combined with efficient pruning and optimization strategies to identify all qualified g-Clusters. Extensive experiments are conducted on real and synthetic datasets. The experimental results show that 1） the algorithm is able to find an amount of co-regulated gene clusters missed by previous models, which are potentially of high biological significance, and 2） the algorithms are effective and efficient, and outperform the existing approaches.     

人工免疫系统在手写体数字识别中的应用

基于生物免疫系统内在的模式识别与记忆能力,通过对手写体数字识别问题的研究,首次将借鉴生物免疫系统发展起来的人工免疫机制和算法引入到该问题中来,定义了相关免疫机制的数学表达,设计了具有学习、记忆、自适应和多样性等特性的人工免疫系统模型,并给出了相应的算法.通过对自采集的手写体数字样本库进行的仿真试验,结果表明用于手写体数字识别的人工免疫系统模型是可靠的,其对应算法可操作性强,只要相关参数选择得当,系统能够得到较好的识别效果,从而体现了人工免疫系统在模式识别领域的优越性和技术潜力.最后对下一步的研究工作进行了展望.     

双波长激光共聚焦生物芯片检测与图像处理

基于共聚焦检测原理,建立了双波长生物芯片检测系统。采用体全息滤光片,利用其反射衍射和带阻滤光特性,无需切换或改变光路,在同一光路中即可实现Cy3与Cy5荧光双波长扫描检测。提出了基于顺序形态变换的生物芯片荧光点阵图像的网格化处理方法,并对Cy3与Cy5荧光试剂点样玻片进行了扫描实验,对实验所得荧光点阵图像进行了网格化处理。实验结果显示,系统的检测灵敏度达到0.1 fluo/μm2,网格化算法简单、有效。     

Identification of Small Regions of Overlap from Copy Number Variable Regions in Patients with Hypospadias

Hypospadias is a common form of congenital atypical sex development that is often associated with other congenital comorbidities. Many genes have been associated with the condition, most commonly single sequence variations. Further investigations of recurrent and overlapping copy number variations (CNVs) have resulted in the identification of genes and chromosome regions associated with various conditions, including differences of sex development (DSD). In this retrospective study, we investigated the DECIPHER database, as well as an internal institutional database, to identify small recurrent CNVs among individuals with isolated and syndromic hypospadias. We further investigated these overlapping recurrent CNVs to identify 75 smallest regions of overlap (SROs) on 18 chromosomes. Some of the genes within these SROs may be considered potential candidate genes for the etiology of hypospadias and, occasionally, additional comorbid phenotypes. This study also investigates for the first time additional common phenotypes among individuals with hypospadias and overlapping CNVs. This study provides data that may aid genetic counseling and management of individuals with hypospadias, as well as improve understanding of its underlying genetic etiology and human genital development overall.     

杜仲预防UVB致人成纤维细胞光老化的作用及基因水平的机制探讨

以UVB辐射体外培养的人成纤维细胞,探讨了杜仲乙醇提取物乙酸乙酯萃取部分(简称杜仲提取物)预防人成纤维细胞光老化的作用,并利用基因芯片对其可能的作用机制进行了探索。实验结果表明,杜仲提取物可以减少UVB对人成纤维细胞活性的影响,具有较好的预防光老化的作用,且基因芯片结果提示这种防护作用可能与提高细胞的抗氧化、DNA修复、抗凋亡有关,同时可能还具有一定促进细胞增殖和预防黑素细胞瘤的作用。     

Activity and Tissue Expression of Tyrosine Phosphatase PTP-MEG2

Protein tyrosine phosphatases(PTPs) are crucial regulators of signal transduction. Among them,PTP-MEG2 is an intracellular enzyme of 593 amino acid residues with a putative lipid-binding domain at the N-terminus. In the present study, we cloned the full-length form of the enzyme and expressed it in E. coli cells as a 6xHis-tagged protein. The majority of the expressed enzyme was found in the inclusion body of E. coli cell extracts.Upon extraction with a buffer containing urea, the recombinant enzyme was purified to near homogeneity on a single Ni-NTA-agarose column. This procedure resulted in the production of over 100 mg of purified recombinant PTP-MEG2 from 1 L E. coli cell culture. The purified protein displayed a single polypeptide band with expected molecular size on SDS-polyacrylamide gel electrophoresis under reducing conditions. Isolated under denatured conditions in urea, the purified enzyme was re-natured by dialyzing against a refolding buffer. The re-natured enzyme effectively dephosphorylated the common PTP substrate para-nitrophenylphosphate with a specific activity of 2000units/mg. Meanwhile, the denatured enzyme was used to immunize a rabbit to produce antibodies. The resulting antiserum had extremely high sensitivity and specificity. When used for Western blot analysis, the anti-serum revealed a wide expression of PTP-MEG2 in many tissues of mice. Together, we developed a highly effective way to purify a large amount of PTP-MEG2 and generated highly sensitive antibodies that can specifically detect endogenous expression of the enzyme in tissues.