传代人淋巴细胞免疫猪的免疫效果初探

目的用传代人淋巴细胞替代人T淋巴细胞进行猪的免疫,用于试制抗人T细胞猪免疫球蛋白(anti-human T lymphocyte porcine immunoglobulin,P-ATG)免疫猪血浆,并评价其免疫效果。方法大量培养传代人淋巴细胞至免疫所需浓度及数量,采用常规猪免疫程序进行2次基础免疫及1次加强免疫,获得免疫猪血浆,用E玫瑰花环形成抑制试验和淋巴细胞毒试验进行效价检测。结果 3批免疫猪血浆的E玫瑰花环形成抑制试验结果均达到1∶1 000(花环形成率均小于对照组平均花环形成率的75%),淋巴细胞毒试验结果均达到1∶500(淋巴细胞死亡率均大于20%),效价均达到《中华人民共和国药典》2015版(三部)中生产用免疫猪血浆的效价标准。结论传代人淋巴细胞可作为人T淋巴细胞的替代免疫原进行猪的免疫,获得了效价合格的免疫猪血浆,用于P-ATG的制备。     

猪白细胞干扰素制备与检定方法

<正>Soloviev等人,连续发表了4篇文章,报导有关猪白细胞干扰素的试验结果,现综述如下: 一、猪白细胞干扰素制备方法 1.制备方法 分离猪白细胞:采血后2～4小时内,加2～3倍容积的0.83％氯化铔溶液、摇匀,4℃静置10分钟。取出,离心沉淀1000转/分10分钟。倒掉上清液(血浆和血红素等)。白细胞用199营养液(其中加10％牛     

明胶亲和层析介质分离纯化猪血浆纤维结合蛋白

目的：研究猪血浆中纤维结合蛋白的分离纯化方法,得到高纯度、高活性的目标蛋白。方法：以明胶亲和层析介质对猪血浆纤维结合蛋白进行分离纯化,考察了不同洗脱方式对纯化结果的影响,对产品进行了纯度、生物活性的测定。结果：通过对纯化工艺条件的摸索,采用含不同浓度尿素缓冲液进行分步洗脱,可以得到纯度为95％以上的产品,回收率达到50．96％;经BHK细胞培养实验证明其具有与人血浆纤维结合蛋白同样的生物活性。结论：采用了较温和的洗脱方式以明胶亲和层析介质对猪血浆纤维结合蛋白进行分离纯化,提高了动物资源的可利用性。     

猪FcγRIII的原核表达及多克隆抗体的制备

目的：构建猪FcγRIII 基因的原核表达载体,诱导表达重组蛋白,制备鼠抗猪 FcγRIII 抗血清。方法：从质粒pTG19-T-FcγRIII中用PCR方法克隆到编码完整猪FcγRIII蛋白分子的基因片段,将其插入到原核表达载体pET-32a中,构建了猪FcγRIII 原核表达载体pET-FcγRIII ,转化大肠杆菌BL21 (DE3) ,IPTG诱导蛋白表达,经尿素洗涤纯化后,以纯化后的融合蛋白FcγRIII-His 为抗原免疫小鼠,获得抗血清。Western blotting、ELISA 法鉴定获得的抗血清,ELISA 结果显示抗体效价为1∶16000,具有高度特异性,免疫印迹结果显示制备的多抗可以与重组猪FcγRIII蛋白特异性结合。结果：成功构建猪FcγRIII原核表达载体,纯化到融合蛋白FcγRIII-His,用纯化的融合蛋白免疫小鼠制备了多克隆抗体,Western blotting、ELISA 法证实多克隆抗体制备成功。结论：成功获得了猪 FcγRIII 多克隆抗体,为进一步研究猪FcγRIII 蛋白的功能奠定了基础。     

猪FcγRⅢ的原核表达及多克隆抗体的制备

目的:构建猪FcγRIII基因的原核表达载体,诱导表达重组蛋白,制备鼠抗猪FcγRIII抗血清。方法:从质粒pTG19-T-FcγRIII中用PCR方法克隆到编码完整猪FcγRIII蛋白分子的基因片段,将其插入到原核表达载体pET-32a中,构建了猪FcγRIII原核表达载体pET-FcγRIII,转化大肠杆菌BL21(DE3),IPTG诱导蛋白表达,经尿素洗涤纯化后,以纯化后的融合蛋白FcγRIII-His为抗原免疫小鼠,获得抗血清。Western blotting、ELISA法鉴定获得的抗血清,ELISA结果显示抗体效价为1∶16000,具有高度特异性,免疫印迹结果显示制备的多抗可以与重组猪FcγRIII蛋白特异性结合。结果:成功构建猪FcγRIII原核表达载体,纯化到融合蛋白FcγRIII-His,用纯化的融合蛋白免疫小鼠制备了多克隆抗体,Western blotting、ELISA法证实多克隆抗体制备成功。结论:成功获得了猪FcγRIII多克隆抗体,为进一步研究猪FcγRIII蛋白的功能奠定了基础。     

猪肺炎支原体Mhp366-N蛋白多克隆抗体的制备及应用

【背景】猪肺炎支原体是猪的一种重要的病原。该菌的研究工具较少,特别是缺少开展其致病机制研究需要的抗体。【目的】制备猪肺炎支原体Mhp366-N蛋白抗体并确定其应用范围和使用时的最佳稀释倍数。【方法】Escherichia coli BL21(DE3)-pET28a(+)-mhp366-N重组菌诱导表达Mhp366-N蛋白并纯化。纯化的蛋白免疫小鼠制备多克隆抗体。用免疫印迹和免疫荧光方法检测猪肺炎支原体AH株感染3D4/21细胞后的Mhp366蛋白,确定2种方法中Mhp366-N多克隆抗体的最佳稀释倍数;之后检测临床采集的猪肺泡巨噬细胞中的猪肺炎支原体;最后以免疫组化试验检测猪肺炎支原体感染的肺细胞。【结果】纯化的Mhp366-N蛋白纯度超过85%,免疫小鼠制备的抗血清效价在1:128 000-1:512 000之间。在免疫印迹试验中Mhp366-N多克隆抗体的最佳工作浓度为1:100 000稀释,免疫荧光试验中Mhp366-N多克隆抗体的工作浓度范围在1:1 000-1:10 000 000,其可用于临床采集的猪肺泡巨噬细胞和细胞系中猪肺炎支原体的检测。免疫组化试验结果显示猪肺炎支原体能够进入猪肺泡巨噬细胞、Ⅰ型和Ⅱ型肺泡上皮细胞。【结论】制备的Mhp366-N多克隆抗体为猪肺炎支原体致病机制研究提供了良好的研究工具。     

神经肽Y存在并作用于血小板

一般认为,血循环中的神经肽Y(NPY)是由神经纤维分泌的。然而,Myers等发现,血循环中的NPY大部分是由血小板释放的,称为免疫活性-NPY(i-NPY)。比较含血小板丰富的血浆(PRP)和含血小板少的血浆(PPP)中i-NPY的量,前者比后者高10倍。i-NPY仅在血小板第二相聚集的不可逆期释放。为了检测i-NPY的化学特性,取大鼠抗凝血制备PRP,用胶原给予最大刺激,引起第二相聚集。聚集后迅速离心的血浆,按常规法处理。同时制备猪NPY标准曲线及大鼠PPP中NPY的曲线。前者仅有单     

慢病毒载体法制备遗传工程猴和小型猪的现状及应用前景展望

较小鼠等啮齿类动物而言,猴和小型猪等大型实验动物在亲缘关系上与人类更为接近,在解剖、生理生化代谢及疾病发病机制等多方面与人类更接近,使它们在复制人类疾病模型,研究疾病发病机制和新药研发等中有无可替代的应用。而制备遗传工程大动物可以更深入地解析人类疾病,并可为器官移植和新药研发提供更充分的实验材料。基于慢病毒介导的转基因方法近几年已越来越多地被用来制备遗传工程猴和小型猪。与传统的原核显微注射方法和体细胞核移植法相比,慢病毒介导的转基因方法转基因效率高,操作更简单。因此,构筑基于慢病毒介导的转基因方法制备遗传工程猴和小型猪的技术平台将对生物医学研究产生巨大推动作用。     

新型冠状病毒假病毒的制备及血浆中和抗体检测

新型冠状病毒（SARS-CoV-2）引发新型冠状病毒肺炎（COVID-19）全球大流行。由于SARS-CoV-2生物安全管理的要求,高效制备获得高滴度的新型冠状病毒假病毒对基于S蛋白研发疫苗、中和抗体、病毒入侵抑制剂药物以及人群血清学调查等十分重要。本研究基于慢病毒系统,对制备新型冠状病毒假病毒过程中的重要参数进行优化,用Western Blot检测假病毒S蛋白和p24蛋白的表达及假病毒包装情况,用荧光素酶报告系统检测假病毒感染入侵效率。利用制备好的假病毒对恢复期血浆的中和抗体水平进行检测。结果显示骨架质粒与野生型Spike质粒为2∶1比例,在转染后48h收集上清为SARS-CoV-2野生型假病毒包装的最佳条件。与野生型相比,恢复期血浆对四种突变株的中和抗体滴度均降低,对B.1.351株中和能力最弱,B.1.617.2株其次,重型患者恢复期血浆对野生型和突变株的中和抗体滴度高于轻型与普通型患者。本研究优化了新型冠状病毒假病毒包装的实验室条件,评估了COVID-19患者恢复期血浆对野生型及四种突变株的中和抗体水平,提示未来对突变株的免疫逃逸进一步加强监测的重要性。     

锌指核酸酶技术制备肌肉生长抑制素基因敲除的五指山小型猪成纤维细胞

敲除猪肌肉生长抑制素(Myostatin, MSTN)基因可能提高猪瘦肉率, MSTN基因敲除猪也可作为相关疾病的动物模型。文章利用锌指核酸酶(Zinc-finger nucleases, ZFNs)技术敲除五指山小型猪胎儿成纤维细胞MSTN基因, 为制备MSTN基因敲除猪奠定基础。ZFNs质粒或编码ZFNs的mRNA均能高效敲除MSTN基因, 使用ZFNs mRNA能直接得到MSTN+/-和MSTN-/-两种基因型的细胞克隆。DNA序列测定与分析发现, 细胞克隆的突变类型多为ZFNs作用靶位点处不大于10 bp的碱基插入或缺失(92.18 %); 氨基酸预测发现, 突变型MSTN基因的终止密码子常常提前出现。将MSTN基因敲除的细胞进行体细胞核移植(Somatic cell nuclear transfer, SCNT)发现, 胚胎体外早期发育潜力与野生型无显著差异, 表明这些细胞可用于后续MSTN基因敲除猪的制备。     

豚鼠腺性膀胱炎模型的建立及尿流动力学的检查

目的探讨豚鼠腺性膀胱炎动物模型的建立方法及尿流动力学检查方法。方法将28只雌性豚鼠分三组：正常对照组、生理盐水对照组和造模组。用膀胱内灌注大肠杆菌的方法制作腺性膀胱炎动物模型,7周后行尿流动力学检查及病理检测。结果尿流动力学检查发现造模组豚鼠储尿期逼尿肌不稳定发生率较正常对照组及生理盐水对照组显著增多（P〈0.001）;正常对照组无腺性膀胱炎病变,生理盐水对照组出现腺性膀胱炎1例,造模组出现7例,造模组与另外两组相比差异有统计学意义（P〈0.05）,正常对照组与生理盐水对照组相比差异无统计学意义（P〉0.05）。结论膀胱内灌注大肠杆菌可导致腺性膀胱炎,这证实了细菌感染是腺性膀胱炎的病因之一,同时为临床上的抗感染治疗提供了理论依据。此方法可用于建立腺性膀胱炎的动物模型。同时确立了腺性膀胱炎动物模型尿流动力学检查方法。     

PURIFICATION OF PHOSPHATE-DEPENDENT PIG BRAIN GLUTAMINASE

Abstract— A procedure for preparing highly purified phosphate-activated glutaminase (EC 3.5.1.2, L-glutamine amidohydrolase) from pig brain is described. The main steps consist of extraction with acetone, followed by sodium sulphate fractionation of the solubilized acetone powder. Thereafter, solubilization by dialysis against a buffer containing tris-HC1, mercaptoethanol, and EDTA, followed by precipitation with phosphate-borate, is repeated twice. The final preparation contains no impurities which can be detected by polyacrylamide gel electrophoresis, isoelectric focusing, and sedimentation equilibrium centrifugation. By the latter method, molecular weight is determined to be 187,000. By polyacrylamide gel electrophoresis in sodium dodecyl sulphate, one protein band with molecular weight 64,000 is found.     

豚鼠脊髓前角神经元线状溶酶体的电镜酶细胞化学研究

本研究采用电镜金属盐法──三偏磷酸酶（ＴＭＰａｓｅ）细胞化学方法探讨了豚鼠脊髓前角神经无线状溶酶体（ＮＬＹ）的存在及其酶细胞化学特点。ＴＭＰａｓｅ是港酶体的标志酶之一。本研究首次以偏磷酸钠代替三偏磷酸钠为ＴＭＰａｓｅ的底物,很好地显示了ＮＬＹ的结构。ＴＭＰａｓｅ反应产物虽高电子密度的黑色铅盐沉淀,不仅分布于圆形溶酶体,也可见于ＮＬＹ,同时,在高尔基复合体的部分板层也有酶活性分布,可能酶是经高尔基复合体加工后输送至溶酶体。ＮＬＹ在神经元胞体,突起及突触中均有分布,并且从胞体到神经终末均有ＮＬＹ和线粒体相贴现象,提示酶是由依赖于线粒体提供运动能量的ＮＬＹ从胞体输送到神经终末,这可能和ＮＬＹ参与神经递质的降解及神经元代谢物质的处理密切相关。     

枫香脂和苏合香的心血管药理学研究

本文比较了苏合香及枫香脂生药、精制枫香脂、枫香脂挥发油的毒性及其对心血管系统的药理作用。结果表明 ,枫香脂毒性小于苏合香。 4种样品均能提高小鼠常压下的心肌耐缺氧能力 ,其中精制枫香脂及枫香脂挥发油的作用最强 ;均可显著降低氯仿诱导的小鼠室颤发生率 ,其中精制枫香脂作用最强 ;对体外血栓干重的抑制率 ,精制枫香脂的作用强于苏合香 ;均可提高冠脉流量 ,其中精制枫香脂及枫香脂挥发油的作用强度及作用维持时间均优于苏合香 ;对离体豚鼠心功能的影响基本相似。提示以精制枫香脂取代苏合香应用于心血管疾病是可能的。     

A protein tyrosine phosphatase inhibitor, sodium orthovanadate, causes parthenogenetic activation of pig oocytes via an increase in protein tyrosine kinase activity.

This study was conducted to determine whether a protein tyrosine kinase (PTK) activity is involved in the initiation of the events that occur at fertilization in pig oocytes. After maturation for 47 h, a 7-h treatment of oocytes with 1 mM sodium orthovanadate, which is an inhibitor of protein tyrosine phosphatase, caused more than 90% pronuclear formation, cortical granule exocytosis, and a decrease in mitogen-activated protein kinase activity. Immunoblotting with an antibody specific for phosphotyrosine showed at least three proteins whose phosphotyrosine contents were significantly increased upon treatment of oocytes with 1 mM sodium orthovanadate. Preincubation of pig oocytes with 50 microM tyrphostin 47, a specific PTK inhibitor, completely blocked the ability of sodium orthovanadate to trigger activation events. In addition, when oocytes were pretreated with the calcium-chelating agent BAPTA-AM, sodium orthovanadate-stimulated pronuclear formation was significantly (P < 0.01) reduced (94.0% vs. 43.1%). These results suggest that PTK may be involved in pig oocyte activation in a calcium-dependent manner and that the stimulation of tyrosine kinase is able to signal a series of intracellular changes that lead to the activation events associated with fertilization.     

Confirmation that catalase is a glycoprotein

Catalases which had been purified from the livers of mouse, rat and guinea pig were subjected to mild periodate oxidation followed by reduction with sodium boro[3H]hydride in order to test for the presence of sialic acid. A radioactively labelled moiety resulted, which behaved as a derivative of N-acetyl neuraminic acid during mild acid hydrolysis, neuraminidase treatment, ion exchange chromatography and paper chromatography. It is concluded that mammalian catalases are glycoproteins, and possess variable amounts of N-acetyl neuraminic acid in their carbohydrate moiety.     

EFFECT OF STIMULATION OF EXCITATORY NERVE TRACT ON RELEASE OF GLUTAMIC ACID FROM OLFACTORY CORTEX SLICES IN VITRO

Abstract— Thin sections prepared from the olfactory cortex of the guinea pig were incubated in a medium containing [¹⁴C]glutamate, and release of radioactive compounds and electrical activity were subsequently examined in the presence of l -cysteate. The postsynaptic potential was almost completely suppressed in the medium containing l -cysteate, whereas the presynaptic potential was unaffected. Repetitive stimulation of the excitatory input of the lateral olfactory tract enhanced release of radioactive glutamate. The facilitatory effect of lateral olfactory tract stimulation increased with increase in stimulus frequency and was dependent on calcium. Release of radioactive gluiamine was not enhanced by lateral olfactory tract stimulation. Phenobarbitone sodium markedly depressed both the postsynaptic potential and the effect of lateral olfactory tract stimulation on glutamate release. These results indicate that stimulation to the lateral olfactory tract enhances liberation of glutamate from the tract nerve terminals.     

Renal physiology of the pig: application of stop-flow

1. The stop-flow method in the pig, with modified kidneys, produced patterns for sodium, chloride, glucose and PAH that were similar to those obtained previously in other animals with simple kidneys. 2. Ethacrynic acid, under the conditions of the experiment, abolished distal sodium and chloride reabsorption in the pig. 3. We conclude that this method, viewed in perspective, could provide useful in situ information about the physiology of modified kidneys where the more difficult micropuncture and in vitro perfusion techniques are impractical.     

Follow-up to the ECVAM prevalidation study on in vitro tests for acute skin irritation. The European Centre for the Validation of Alternative Methods Skin Irritation Task Force report 2

The European Centre for the Validation of Alternative Methods (ECVAM) Skin Irritation Task Force was established in 1996, to review the status of the development and validation of alternative tests for skin irritation and corrosion, and to identify appropriate non-animal tests for predicting human skin irritation that were sufficiently well-developed to be prevalidated and validated by ECVAM. The EpiDerm method, based on a reconstituted human skin model, was proposed as being sufficiently well advanced to enter a prevalidation (PV) study. Based on a review of test protocols, prediction models (PMs), and data submitted by test developers on ten specified chemicals, with 20% sodium lauryl sulphate as a reference standard, the task force recommended the inclusion of four other tests: EPISKIN and PREDISKIN, based on reconstituted human epidermis or on human skin; the non-perfused pig-ear test, based on pig skin; and the skin integrity function test (SIFT), with ex vivo mouse skin. The prevalidation study on these methods was funded by ECVAM, and took place during 1999-2000. The outcome of the PV study was that none of the methods was ready to enter a formal validation study, and that the protocols and PMs of the methods had to be improved in order to increase their predictive abilities. Improved protocols and PMs for the EpiDerm and EPISKIN methods, the pig ear test, and the SIFT were presented at an extended Task Force meeting held in May 2001. It was agreed that, in the short term, the performance of the revised and harmonised EpiDerm and EPISKIN methods, as well as the modified SIFT, should be evaluated in a further study with a new set of 20 test chemicals. In addition, it was decided that the SIFT and the pig ear test would be compared to see if common endpoints (transepidermal water loss, methyl green-pyronine stain) could be identified.     

THE EFFECTS OF SERUM ON SPERMATOZOA

A spermicidal factor was found in fresh human, bovine, rabbit, guinea pig, and rat sera. It kills the spermatozoa of its own species (except in the case of human serum) and the sperms of other species. It was unstable, thermolabile, and of large molecular size. It was present in limited quantity in the fresh serum and could be used up by a definite number of spermatozoa. It could be destroyed by sodium citrate, by Seitz filtration, by trypsin, and by snake venom. This factor was not present in tissue extracts and various plasma protein fractions. The strength or concentration of this factor varies in different individuals and in different species. This factor has several characteristics similar to those of complement.