交叉中和血清1型和血清3型鸭甲肝病毒单克隆抗体的制备与鉴定

鸭甲型肝炎（duck hepatitis A,DHA）是危害养鸭业的主要疾病之一,是一种雏鸭的急性、高度致死性传染病。本研究旨在研制一种对我国流行的鸭甲型肝炎血清1型（DHAV-1）和血清3型（DHAV-3）病毒具有交叉中和作用的单克隆抗体（monoclonal antibody,McAb）。人工合成12条DHAV-1和DHAV-3的VP1蛋白共有的抗原表位肽,分别与钥孔血蓝蛋白（keyhole limpet hemocyanin,KLH）载体偶联作为免疫抗原,免疫BALB/c小鼠制备McAb。通过检测McAb与DHAV-1和DHAV-3的交叉反应性,测定McAb对病毒增殖的抑制效率、中和效价以及攻毒保护率等来筛选McAb。本研究共获得了12株稳定分泌McAb的杂交瘤细胞株,其中6株（C1、C4、C7、C12、C13、C16）分泌的McAb同时与DHAV-1和DHAV-3发生特异性交叉反应; C1、C4、C7、C16可以抑制DHAV-1和DHAV-3在鸭胚中的增殖,抑制率在75.34%～100.00%不等;对DHAV-1和DHAV-3的中和效价：C1（1：3&1：5）、C4（1：3&1：3）、C7（1：10&1：11）和C16（1：9&1：9）;C7和C16对DHAV-1和DHAV-3攻毒雏鸭的保护率较高,分别为“70%、80%”和“100%、60%”。本研究成功研制出对DHAV-1和DHAV-3具有交叉中和活性的McAb 4株,其中2株对病毒的感染具有良好的预防效果,为DHA的防控提供了新材料和新思路。     

Swine viscera preservation in hypersaturated salt solution after alcohol fixation as a preparation method for educational purposes

The use of live animals for educational purposes is an old practice that is still employed in teaching and research institutions. However, there are several objections to this practice, whether for ethical or humanitarian reasons. Surgical techniques teaching using anatomical pieces and/or preserved cadavers promotes greater learning efficiency, provides exercise repetition and increases the confidence and satisfaction of the students when compared to the use of live animals. The current work aimed to analyse the feasibility of using fresh swine urinary bladder and small intestines (jejunum), obtained from slaughterhouses, fixed in 99.8% ethyl alcohol (EA) and preserved in sodium chloride hypersaturated solution (SCHS) at 30%, for 7, 14 and 21 days, as an alternative method for surgical skills training (SST). Swine viscera, fixed in EA and preserved in SCHS, presented a realistic appearance, absence of odour and maintained the viable morphological characteristics during the performance of the operative techniques. Preservation solutions had low cost, were easy to acquire and did not offers risks to human health. Therefore, urinary bladders and small intestines fixed in 99.8% EA for 30 days and maintained in 30% SCHS at different periods were demonstrated as a good viable option as a preservation method for surgical skills training.     

水溶性烯丙孕素-磺丁基-β-环糊精包合物的制备及表征

旨在提高烯丙孕素原药在水中的溶解度,提高烯丙孕素原药的生物利用度,使用磺丁基-β-环糊精对烯丙孕素原药进行包合。本试验使用冷冻干燥法来制备烯丙孕素-磺丁基-β-环糊精包合物,以此来解决烯丙孕素在临床使用中因其水不溶性而受到限制及其使用后生物利用度低的问题,进而拓宽烯丙孕素的药用途径。通过傅里叶变换红外光谱法、热重分析法和显微镜成像法对所制得的烯丙孕素包合物进行表征,并用溶解度法对包合物进行溶解度测定。以包合物的收率和包合率为评价指标,筛选最优的包合物制备条件。结果显示：经筛选,包合物的最佳制备条件：在55℃,ALT与SBE-β-CD的投料摩尔比为1：6,包合时间为5 h,溶液pH为8,溶剂量为15 mL（当以ALT投药量为0.1 g时计）;以最优制备条件制备所得的包合物中ALT的平均包合率为（90.90±1.80）%（n=3）,平均载药量为（4.30±2.30）%（n=3）,包合物的收率为（93.19±1.67）%。经傅里叶变换红外光谱法、热重分析法和显微镜成像法对所制得的包合物进行表征,可证实成功制得包合物。溶解度法试验结果表明,形成包合物后,其溶解度较烯丙孕素原药提高了988.86倍,且该包合物稳定性好,可满足不同剂型的要求。磺丁基-β-环糊精对烯丙孕素原药具有较好的增溶作用,达到了增加药物溶解度的目的,且该制备方法简单,条件温和,易于产业化生产,有利于烯丙孕素的进一步开发利用。     

参考群筛选方法及规模对基因型填充准确性的影响

为探究基于A矩阵期望遗传关系最大化（maximizing the expected genetic relationship for matrix A,RELA）、基于A矩阵目标群体遗传方差最小化（minimized the target population genetic variance for matrix A,MCA）、平均亲缘关系最大化（the highest mean kinship coefficients,KIN）、随机选择（random selection,RAN）、共同祖先筛选（common ancestor,CA）等不同参考群筛选方法及参考群规模对基因型填充准确性的影响。本研究使用矮小型黄羽肉鸡作为试验群体,采用鸡600K SNP芯片（Affymetrix Axion HD genotyping array）进行基因分型,测定435羽子代公鸡45、56、70、84、91日龄体重。利用Beagle软件将低密度SNP芯片填充为高密度SNP芯片数据,比较不同参考群筛选方法、参考群规模对基因型填充准确性的影响,以及填充芯片基因组预测准确性。结果表明,使用Beagle 4.0结合系谱信息进行填充效果最佳,其次为Beagle 4.0,而Beagle 5.1填充效果最差。使用MCA方法筛选参考群进行基因型填充准确性最高,使用RAN方法筛选参考群进行基因型填充准确性最低,MCA、RELA、CA 3种方法基因型填充准确性差别较小。相比其他方法,使用MCA方法筛选个体作为参考群将低密度SNP芯片填充至高密度SNP芯片进行基因组选择的预测准确性较高,与真实高密度SNP芯片的基因组预测准确性相差甚微。随着参考群规模增大,基因型填充准确性也随之增加,但增速逐渐下降,最后趋于平缓。综上所述,可以通过参考群筛选方法构建参考群以及控制参考群规模,以保证基因型填充和基因组预测准确性并节省成本,本研究为基因型填充在畜禽遗传育种中的应用提供技术参考。     

Molecular characterization of Ascaridia galli from Bangladesh and development of a PCR method for distinguishing A. galli from Heterakis spp.

We analyzed the nuclear ribosomal internal transcribed spacer (ITS) 1 and ITS2 sequences for Bangladesh isolates of Ascaridia galli, and we determined that the sequences were unreliable as molecular markers for distinguishing A. galli from other Ascaridia species, because the sequences showed high identity with that of A. columbae. However, the ITS1 sequences were available for designing PCR primers distinguishable between Ascaridia galli and Heterakis spp. Bangladesh isolates of A. galli constituted a monophyletic clade along with other geographical isolates in the cytochrome c oxidase subunit I (COI) phylogenetic tree, however, we could not clarify the phylogenetic relationships between A. galli and other Ascaridia spp., because their available sequences in GenBank were very few. The developed PCR method using DNA from A. galli and Heterakis spp. eggs would enable differential diagnosis of the individual infections in the future.     

Relationship between anti-insulin antibody production and severe insulin resistance in a diabetic cat

A 5-year-old castrated male domestic shorthair cat was diagnosed with diabetic ketoacidosis and severe insulin resistance. Although the conventional treatment for diabetic ketoacidosis was provided, the cat required frequent hospitalization because of severe dehydration and repeated diabetic ketoacidosis. We detected anti-insulin antibodies for human in this cat. Serum insulin-binding IgG levels were markedly elevated compared with those in healthy cats and other diabetic cats. We initiated prednisolone to suppress the effects of anti-insulin antibodies. After initiation of prednisolone, the cat was gradually recovered with increasing activity and appetite. Furthermore, satisfactory glycemic control was achieved with combined subcutaneous injection of insulin detemir and insulin degludec.     

Recovery of sperms bearing X chromosomes with different concentrations of magnetic nanoparticles in ram

Pre-conceptual sex selection is still a highly debatable process whereby X and Y chromosome bearing spermatozoa are isolated before oocyte fertilization. Recently, magnetic nanoparticles (MNP) have been used to determine X and Y chromosomes bearing spermatozoa as a result of searching for a cheap, highly efficient method using non-toxic materials. This study aimed to recover the sperm bearing X chromosomes in ram with different concentrations of MNP and then evaluate the success of this method using polymerase chain reaction (PCR). Ram sperms were divided into four groups, treated with 0 (control), 50, 100 and 200 μg/ml MNP, respectively. MNP was used to restore sperm cells bearing X chromosomes. Upon recovery, the PCR was performed to identify the X and Y sperms, Methyl ThiazoleTetrazolium (MTT), to assess MNP toxicity and sperm viability and acridine orange (AO) to evaluate sperm DNA integrity. The results of PCR revealed that the treatment of spermatozoa- bearing X chromosomes with 50 μg/ml MNP had the highest effects on the recovery of X sperm rather than the other concentrations of MNP. However, the concentrations of MNP did not have any toxic effects on spermatozoa, sperm viability and, DNA integrity, but the high concentration of MNP (200 μg/ml) significantly reduced DNA integrity. According to MTT and AO results, the concentrations of MNP used in this study had no toxic effects on spermatozoa and did not reduce the sperm viability and DNA integrity, except that 200 μg/ml MNP significantly reduced DNA integrity.     

Production of monoclonal antibody against recombinant bovine sex-determining region Y (SRY) and their preferential binding to Y chromosome-bearing sperm

Separation of X and Y chromosome-bearing sperm is an appropriate method for the selection of desired sex of offspring to increase the profit in livestock industries. The purpose of this study was the production of a monoclonal antibody against recombinant bovine sex-determining region Y protein for separation Y sperm. The hybridoma cells from splenocytes of immunized female's balb/C mice and Sp2/0 cells were made. The binding affinity of our monoclonal antibody (mAbSRY2) was compared with mouse monoclonal SRY-15. The Western blot method indicated that mAbSRY2 successfully detected the rbSRY protein. The specificity and sensitivity of mAbSRY2 is comparable to SRY-15 commercially ones. The SRY gene in 100% of bull semen contains the Y chromosome that had the strongest binding affinity to mAbSRY2 was synthesized. In other words, the binding affinity of semen contains the X sperms near the negative control. In general, this immunological method can help to separate X from Y sperms. However, the mAbSRY2 is bind to Y-bearing sexed sperm, but in the future; the sexed sperms need to apply in farms.     

厄贝沙坦及其复方制剂中痕量杂质N-亚硝基-N-甲基-4-氨基丁酸残留方法学研究

采用超高效液相色谱-三重四级杆质谱(UPLC-MS/MS)建立厄贝沙坦及其复方制剂中N-亚硝基-N-甲基-4-氨基丁酸(NMBA)的测定方法。采用Waters ACQUITY UPLC○RHSS T3色谱柱进行分离,以0.005 mol/L甲酸铵(用甲酸调pH值至3.0)为流动相A,甲醇为流动相B,梯度洗脱,质谱检测器,流速为0.3 mL/min。结果显示NMBA的线性范围为0.156 6~3.132 6 ng/mL,相关系数r>0.99,方法检出限浓度为0.047 0 ng/mL(相当于样品浓度0.01μg/g),定量限浓度为0.156 6 ng/mL(相当于样品浓度0.03μg/g),样品加标回收率在80%~130%。说明该方法可以快速、简便、灵敏、准确地测定厄贝沙坦及其复方制剂中NMBA含量。     

生物酶法提取中草药活性成分研究进展

对酶提方法的原理、酶类选择依据、酶提工艺,以及酶提法应用现状进行了综述,旨在为同类研究提供技术参考。中草药具有广泛的应用前景和开发潜力,为提高中药疗效,方便临床用药,生产中常需要对中草药进行提取,然而传统的提取方法较为繁杂。随着科技进步,一些现代中药提取方法不断涌现。酶提方法是一种应用酶工程技术提取中药的现代中药提取方法,该方法具有高效、无毒、反应条件温和等优点,是一种发展潜力较大的中药提取方法。