蛇毒金属蛋白酶抑制剂抗小鼠黑色素瘤肺转移

探讨蛇毒金属蛋白酶抑制剂BJ46a抗肿瘤侵袭转移作用。以BJ46a为目的基因,选择杆状病毒表达系统生产重组BJ46a蛋白,ProBondTM亲和层析纯化后处理黑色素瘤细胞B16,经尾静脉接种C57BL/6小鼠,建立B16细胞实验性肺转移模型,20天后以颈椎脱臼法处死小鼠,观察肺组织病理学变化。结果表明：不同浓度重组BJ46a蛋白处理组肺部转移瘤数分别为1.1±0.83、0.9±0.7,明显低于对照组（6.3±3.00,P<0.001）和空白对照组（10.7±5.73,P<0.001）,光镜下对照组肿瘤病理学征象明显,而重组BJ46a蛋白处理组瘤结节小。研究结果首次证明了重组BJ46a蛋白能抑制小鼠黑色素瘤细胞的侵袭转移,为其作为抗肿瘤侵袭转移药物的进一步研制和开发应用提供理论依据和前提条件。探讨蛇毒金属蛋白酶抑制剂BJ46a抗肿瘤侵袭转移作用。以BJ46a为目的基因,选择杆状病毒表达系统生产重组BJ46a蛋白,ProBond™ 亲和层析纯化后处理黑色素瘤细胞B16,经尾静脉接种C57BL/6小鼠,建立B16细胞实验性肺转移模型,20天后以颈椎脱臼法处死小鼠,观察肺组织病理学变化。结果表明：不同浓度重组BJ46a蛋白处理组肺部转移瘤数分别为1.1±0.83、0.9±0.7,明显低于对照组（6.3±3.00,P<0.001）和空白对照组（10.7±5.73,P<0.001）,光镜下对照组肿瘤病理学征象明显,而重组BJ46a蛋白处理组瘤结节小。本研究结果首次证明了重组BJ46a蛋白能抑制小鼠黑色素瘤细胞的侵袭转移,为其作为抗肿瘤侵袭转移药物的进一步研制和开发应用提供理论依据和前提条件。     

α-环状糊精葡萄糖基转移酶的固定化及其性质研究

研究了一种α-环糊精葡萄糖基转移酶的固定化和固定化酶的性质。通过对戊二醛浓度、酶量和交联时间各单因素的考察,确定了最佳的固定化条件。与游离酶相比,以DEAE纤维素为载体的固定化酶最适pH向酸性偏移,最适温度不变,pH稳定性和热稳定性都有所提高。在40℃、150r/min下反应3h,转化率可以达到32%。固定化酶可以连续使用4次以上。固定化酶在4℃、5mmol/L CaCl2溶液里保存18d,还剩余80%以上的活力。     

α-2,6唾液酸转移酶(ST6Gal1)的cDNA克隆、原核表达及生物活性研究

从HepG2细胞中提取总RNA,经RT-PCR扩增α-2,6唾液酸转移酶基因,构建于pMD-18T克隆载体中,经序列测定后与GenBank中报道的已知序列比对完全一致,再将已知目的序列插入原核表达载体pET-28a(+)中。将构建正确的原核表达载体转化表达受体菌BL21（DE3）中,IPTG诱导其进行表达并鉴定目的蛋白,对鉴定正确的目的蛋白复性后经Ni2 +亲和层析柱纯化获得高纯度的α-2,6唾液酸转移酶蛋白。然后用霍乱弧菌神经氨酸酶处理健康鸡红细胞,清除鸡红细胞表面所有类型的唾液酸寡糖链;再用表达的α-2,6唾液酸转移酶以 CMP-唾液酸作为底物在霍乱弧菌神经氨酸酶处理的鸡红细胞表面标记上SAα2,6 Gal受体。将标记有SAα2,6Gal受体的鸡红细胞应用微量血凝试验和流式细胞仪进行检测,以验证所表达蛋白的生物活性。结果表明,原核表达的α-2,6唾液酸转移酶具有较好的生物活性。     

NASBA(依赖核酸序列的扩增)及其在病毒检测中的应用

NASBA(nucleic acid sequence based amplification)即依赖核酸序列的扩增技术,是一种扩增RNA的新技术,是由一对引物介导的、连续均一的、体外特异核苷酸序列等温扩增的酶促反应过程。反应在42℃进行,可以在2h左右将模板RNA扩增约109~12倍,不需特殊的仪器。NASBA已经广泛应用于细菌、病毒等多种病原微生物的检测,就NASBA的技术原理及其在病毒检测中的应用作一综述。     

EGFP-LacZ双报告基因真核表达载体的构建及体外表达

构建(β-半乳糖苷酶与增强型绿色荧光蛋白(EGFP)双报告基因的真核表达载体,并在真核细胞中表达。采用PCR方法从质粒pLenti6/V5-GW/LacZ 中获取LacZ全基因,与pEGFP-C1重组后构建真核表达载体pEGFP-C1-LacZ。该重组质粒经PCR和酶切方法鉴定后,在脂质体介导下转染293FT细胞株及鸡胚成纤维细胞,通过荧光观察和组织化学方法检测Egfp和LacZ基因的表达。结果表明,LacZ基因被克隆到pEGFP-C1,成功构建了双报告基因真核表达载体。该重组质粒转染后的细胞呈现绿色荧光,组织化学方法检测到呈蓝色的细胞,表明两个报告基因均能在细胞内正确表达。     

人Trim5α嵌合体蛋白与HIV-1gag蛋白体外分子间相互作用的鉴定

目的:表达和纯化人TRIM5α嵌合体蛋白［TRIM5α-H(R328-332)］,并探讨该蛋白和HIV-1gag间的相互作用。方法:将构建的TRIM5α嵌合体pET-28a-TRIM5α-H(R328-332),转化大肠杆菌BL21(DE3) ,获得重组表达质粒pETTRIM5α-H(R328-332),30℃下经IPTG诱导,该融合蛋白在大肠杆菌BL21(DE3)中表达。获得的重组的蛋白再经过纯化,SDS-PAGE分析重组蛋白,并用免疫共沉淀技术和ELISA等检测重组蛋白与HIV-1 gag间的相互作用。结果:构建的重组质粒在大肠杆菌中获得表达,重组TRIM5α-H(R328-332)蛋白经纯化复性后,通过免疫共沉淀和ELISA等技术,证明TRIM5α-H(R328-332)蛋白能够与HIV-1gag间的相互作用。 结论:在大肠杆菌表达系统中成功表达了重组TRIM5α-H(R328-332)蛋白,并且证实其在体外与HIV-1gag有结合作用。[摘要]　目的:表达和纯化人Trim5α嵌合体蛋白[Trim5α H(R328-332)],并探讨该蛋白和HIV-1gag间的相互作用。方法:将构建的Trim5α嵌合体pET 28a Trim5α H(R328-332),转化大肠杆菌BL21(DE3) ,获得重组表达质粒pETTrim5α H(R328-332),30℃下经IPTG诱导,该融合蛋白在大肠杆菌BL21(DE3)中表达。获得的重组的蛋白再经过纯化,用SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)分析重组蛋白,并用免疫共沉淀技术、His pull down和ELISA等检测重组蛋白与HIV-1gag间的相互作用。结果:构建的重组质粒在大肠杆菌中获得表达,经纯化后的重组Trim5α H(R328-332)蛋白纯度可达90%以上。通过免疫共沉淀、GST pull down和ELISA等技术,证明Trim5α H(R328-332)蛋白能够与HIV-1gag间的相互作用。 结论:本实验在大肠杆菌表达系统中成功表达了重组Trim5α H(R328-332)蛋白,并且证实其在体外与HIV-1gag有结合作用。     

龟纹瓢虫广东种群和北京种群的耐热性比较研究

本文以龟纹瓢虫Propylea japonica (Thunberg)的广东和北京两地区种群为材料,对其进行了在不同温度（26℃～36℃）下的发育历期、存活率、种群性比、产卵量的试验观察及对蛹重、幼虫致死高温、亚致死高温预处理、可溶性蛋白质含量的测定等。结果显示：（1）34℃以上温度对两种群龟纹瓢虫的各世代历期均产生了不同程度的影响。广东种群,34℃下的4龄幼虫、成虫产卵前期和世代历期开始延长,在36℃时除1龄幼虫和蛹外,其它虫态的发育历期均比34℃时的显著延长;北京种群,在36℃时,2龄和4龄幼虫的发育历期开始表现为显著延长。雄性比例,在26℃～36℃范围内随着温度上升而增大,至36℃时,两种群成虫均为雄性。从产卵量看,两种群在34℃下急剧下降。（2）在供试条件下,两种群各龄期的存活率,在36℃下开始明显下降,其中对低龄幼虫和卵期影响最大。广东种群卵的孵化率从26℃时的64.3%下降到34℃时的42.3%, 36℃下的则降为20.7%,下降了67.8％;北京种群卵的孵化率从26℃处理的58.0%, 下降到34℃处理的44%,36℃处理仅为12.3%,下降了78.8％。（3）广东种群和北京种群各龄幼虫致死温度相同,分别为1、2龄幼虫44℃、3龄幼虫45℃、4龄幼虫46℃。经39℃亚致死高温预处理150 min后,广东种群和北京种群4龄幼虫在46℃下的存活率分别提高了72%和43.5%。（4）在26℃～34℃之间,随着温度的上升,成虫和4龄幼虫体内可溶性蛋白的含量上升。在常温下生长的4龄幼虫和成虫经39℃、41℃和43℃热激1 h后,4龄幼虫体内可溶性蛋白含量上升,成虫体内可溶性蛋白含量却下降。广东种群4龄幼虫和成虫经39℃热激后可溶性蛋白含量最高,而北京种群经43℃热激后含量最高。本文结果初步明确了龟纹瓢虫对较高温度的忍耐性,发现在36℃下龟纹瓢虫广东种群和北京种群的生长发育和繁殖会受到严重阻碍,对其发育历期、性比、产卵量、存活率等均有不良影响;广东种群和北京种群在31℃下3龄幼虫的发育历期、36℃下世代存活率、经过亚致死高温预处理后的耐热性、热激后虫体内蛋白含量等均存在显著差异。, -bidi-font-family: 'Times New Roman'; mso-bidi-font-size: 12.0pt; mso-font-kerning: 1.0pt; mso-ansi-language: EN-US; mso-fareast-language: ZH-CN; mso-bidi-language: AR-SA">热激1 h后,4龄幼虫体内可溶性蛋白含量上升,成虫体内可溶性蛋白含量却下降。广东种群4龄幼虫和成虫经39℃热激后可溶性蛋白含量最高,而北京种群经43℃热激后含量最高。本文结果初步明确了龟纹瓢虫对较高温度的忍耐性,发现在36℃下龟纹瓢虫广东种群和北京种群的生长发育和繁殖会受到严重阻碍,对其发育历期、性比、产卵量、存活率等均有不良影响;广东种群和北京种群在31℃下3龄幼虫的发育历期、36℃下世代存活率、经过亚致死高温预处理后的耐热性、热激后虫体内蛋白含量等均存在显著差异。, -bidi-font-size: 12.0pt; mso-font-kerning: 1.0pt; mso-ansi-language: EN-US; mso-fareast-language: ZH-CN; mso-bidi-language: AR-SA; mso: 'Times New Roman'">热激1 h后,4龄幼虫体内可溶性蛋白含量上升,成虫体内可溶性蛋白含量却下降。广东种群4龄幼虫和成虫经39℃热激后可溶性蛋白含量最高,而北京种群经43℃热激后含量最高。本文结果初步明确了龟纹瓢虫对较高温度的忍耐性,发现在36℃下龟纹瓢虫广东种群和北京种群的生长发育和繁殖会受到严重阻碍,对其发育历期、性比、产卵量、存活率等均有不良影响;广东种群和北京种群在31℃下3龄幼虫的发育历期、36℃下世代存活率、经过亚致死高温预处理后的耐热性、热激后虫体内蛋白含量等均存在显著差异。, -bidi-font-family: 'Times New Roman'; mso-bidi-font-size: 12.0pt; mso-font-kerning: 1.0pt; mso-ansi-language: EN-US; mso-fareast-language: ZH-CN; mso-bidi-language: AR-SA">热激1 h后,4龄幼虫体内可溶性蛋白含量上升,成虫体内可溶性蛋白含量却下降。广东种群4龄幼虫和成虫经39℃热激后可溶性蛋白含量最高,而北京种群经43℃热激后含量最高。本文结果初步明确了龟纹瓢虫对较高温度的忍耐性,发现在36℃下龟纹瓢虫广东种群和北京种群的生长发育和繁殖会受到严重阻碍,对其发育历期、性比、产卵量、存活率等均有不良影响;广东种群和北京种群在31℃下3龄幼虫的发育历期、36℃下世代存活率、经过亚致死高温预处理后的耐热性、热激后虫体内蛋白含量等均存在显著差异。     

RNAi介导的棉铃虫氨肽酶N基因Haapn1和钙粘蛋白基因Ha_BtR沉默对Cry1Ac毒力的影响

氨肽酶N（aminopeptidase N, APN）和钙粘蛋白（cadherin）是存在于鳞翅目昆虫中肠刷状缘膜囊（brush border membrane vesicles, BBMV）上Bt毒素Cry1A的受体。本实验将棉铃虫Helicoverpa armigera氨肽酶N1基因Haapn1和钙粘蛋白基因Ha_BtR双链RNA（dsRNA）注入棉铃虫4龄幼虫体内, 以研究这两种受体基因沉默后对Cry1Ac毒力的影响。结果表明: 注射dsRNA（1 μg/头）进行基因沉默后, Haapn1 mRNA表达量比注射缓冲液（elution solution, ES）的对照下降了30%~49%, Ha_BtR mRNA表达量下降了30%~37%。注射Haapn1 dsRNA的幼虫在40和70 μg/cm² Cry1Ac活化毒素下的死亡率显著低于注射ES的幼虫, 而在 100 和 170 μg/cm² Cry1Ac原毒素处理下两者死亡率无显著差异; Cry1Ac活化毒素以及原毒素对注射Ha_BtR dsRNA幼虫与注射ES幼虫的毒力均无显著差异。当同时注射Haapn1及Ha_BtR dsRNA后, 干扰后的幼虫对Cry1Ac活化毒素和原毒素的敏感性均显著下降。本研究进一步证明了棉铃虫Haapn1和Ha_BtR均是Bt毒素Cry1Ac的功能受体, 这两种受体蛋白共同参与Cry1Ac的毒杀作用过程。该结果也提示, Haapn1或Ha_BtR基因产生突变都可能导致棉铃虫对Cry1Ac产生抗性。     

异色瓢虫成虫体型及体内脂肪含量对其耐寒能力的影响

为明确异色瓢虫Harmonia axyridis (Pallas)成虫体型及体内脂肪含量对其耐寒能力的影响, 本研究在2种发育温度(18℃和25℃)下通过表型可塑性诱导获得了不同体型的异色瓢虫成虫, 测定了成虫体型大小、体内脂肪含量、过冷却点(supercooling point, SCP)及其在恒定低温(constant low temperature, CLT)和变温(fluctuating thermal regime, FTR)下的存活率。结果表明: 较低发育温度(18℃)下的成虫体型比25℃下的明显大(P<0.01), 体内脂肪含量显著高于25℃下的(P<0.01); 且两2种发育温度下成虫体内脂肪含量与虫体干重呈较好的正相关关系。由SCP频次分布图可知, 饲养在18℃下的成虫SCP主要集中在-8 ~ -6℃范围内, 饲养在25℃下的成虫SCP主要集中在-10 ~ -9℃范围内; 且成虫体型大小与过冷却能力呈现负相关关系。经过相同时间低温暴露后成虫的存活率按下列顺序降低: FTR18℃＞FTR25℃＞CLT18℃＞CLT25℃。结果表明低温暴露过程中脂肪贮存对异色瓢虫成虫存活是非常重要的, 但是冷伤害是影响存活的基本因素。     

温度对马铃薯甲虫生长发育的影响

为了进一步明确温度对马铃薯甲虫Leptinotarsa decemlineata (Say)生长发育的影响, 在恒温条件, 研究了温度对马铃薯甲虫实验种群生长发育的影响。结果表明：温度对马铃薯甲虫各虫态的发育历期、存活率及种群的繁殖力有显著的影响。发育历期随温度的升高而缩短, 发育速率与温度呈显著的正相关。马铃薯甲虫世代存活率由大到小的顺序为27℃＞23℃＞19℃＞31℃＞15℃。27℃时成虫的产卵量最高, 单雌平均卵量为729.7粒;其次为23℃, 单雌平均卵量为639.2粒。并测得马铃薯甲虫卵、1龄幼虫、2龄幼虫、3龄幼虫、4龄幼虫、幼虫期、蛹期及全世代的发育起点温度分别为9.14, 10.50, 8.17, 10.28, 9.04, 9.59, 10.23和10.90℃, 有效积温分别为73.26, 43.22, 39.23, 34.05, 161.97, 273.02, 100.38和542.58日·度。据此认为温度对马铃薯甲虫实验种群的生长发育、存活和繁殖影响明显。     

抗麻痹性贝毒素GTX2,3单克隆抗体的制备及特性分析

制备抗麻痹性贝毒GTX2,3单克隆抗体。利用醛化法将GTX2,3与载体牛血清白蛋白(BSA)偶联,制备完全抗原。免疫小鼠,取小鼠脾细胞与Sp2/0细胞融合。GTX2,3与钥孔血蓝蛋白(KLH)偶联作为检测抗原,用间接ELISA法筛选阳性克隆株。将筛选的阳性细胞株制备腹水。获得三株稳定分泌抗GTX2,3单克隆抗体的杂交瘤细胞株F4、F10、G9。间接ELISA法检测F10细胞株腹水抗体效价为1.4×10^-5。半抗原GTX2,3与载体蛋白偶联后,作为免疫原,可制备高滴度的抗GTX2,3抗血清和单克隆抗体。该抗体对于藻毒素具有高特异性和高亲和力,可用于污染海产品的麻痹性贝毒的检测。     

玉米赤霉烯酮单克隆抗体和免疫酶技术研究

采用蛋白质连接技术合成玉米赤霉烯酮抗原,免疫Balb/c鼠,通过淋巴细胞杂交瘤技术建立六株分泌抗玉米赤霉烯酮的单克隆抗体杂交瘤细胞株。间接酶联免疫吸附试验测定细胞上清抗体效价为1:2084(4H8)、1:256(6H9、4H3、2H5、2C8)、1:16(3F10);腹水抗体效价为10~9(4H3、4H8)、10~8(2H5)、10~7(6H9)、10~5(3H10)。竞争间接酶联免疫吸附试验测定六株单克隆抗体对玉米赤霉烯酮的敏感度为0.3—0.8ng/ml。六株抗体与玉米亦霉烯醇的交叉反应率为1.3—9.0％。六株单克隆抗体均属IgG类。细胞体外传代培养和冻存复苏后分泌抗体稳定。纯化抗体在37℃保存12天稳定,-30℃保存90天抗体滴度不变。用该抗体建立竞争间接酶联免疫吸附试验检测掺合玉米赤霉烯酮的玉米、小麦、饲料,平均回收率分别为105％、90％、103％,平均批间变异系数为5.8％、2.8％、6.8％,批内变异系数为3.8％、12.7％、15.7％。样品中玉米赤霉烯酮掺合量与竞争间接酶联免疫吸附试验检出量有良好相关性(r≥0.9996)。     

牛血清白蛋白单克隆抗体杂交瘤细胞株的建立及鉴定

用牛血清白蛋白(BSA)免疫BALB/c小鼠,取其脾细胞与小鼠骨髓瘤细胞SP2/0进行融合,培养上清经过双抗体夹心法检测初步筛选分泌鼠IgG的杂交瘤细胞,将此种杂交瘤细胞注射小鼠产生的腹水用间接ELISA法筛选,获得4株能稳定分泌抗BSA单克隆抗体的杂交瘤细胞株,分别命名为2A5、3A3、3G6、4A8。鉴定结果显示,2A5细胞分泌IgG2a/κ,其余3株细胞分泌IgG1/κ;纯化后4株腹水单抗的纯度达90%以上,对BSA的ELISA滴度均可达到1∶100000以上;4株单抗均不与人以及马、猪、羊、兔、豚鼠等血清发生交叉反应;W estern B lotting试验证明4株单抗均识别分子量为68000的BSA;用间接ELISA法测定4株单抗相对亲和力及相对敏感度大小依次为3A3>2A5>3G6>4A8;杂交瘤细胞株连续培养3个月以及冻存半年后复苏,细胞生长良好,杂交瘤细胞分泌的抗体效价稳定。     

CD95 (Fas) ligand-expressing vesicles display antibody-mediated, FcR-dependent enhancement of cytotoxicity

Bioactive Fas ligand (FasL)-expressing vesicles were generated (vesicle preparation, VP) from two cell lines overexpressing FasL. The effect of NOK-1 anti-FasL mAb (mouse IgG1) on the cytotoxicity of FasL VP against various targets was determined. At high concentrations (1-10 microg/ml), NOK-1 inhibited the cytotoxicity. By contrast, NOK-1 in the dose range of 1-100 ng/ml significantly enhanced cytotoxicity against the FcR(+) LB27.4, M59, and LF(+) targets, but not the FcR(-) Jurkat and K31H28 hybridoma T cell targets. The ability to enhance FasL VP-mediated cytotoxicity could be blocked by the FcR-specific mAb 2.4G2. Enhancement was also observed with FcR(+) A20 B lymphoma but not with the FcR(-) A20 variant. Enhancement of FasL VP cytotoxicity was observed with five IgG anti-FasL mAbs, but not with an IgM anti-FasL mAb. Inhibition was observed with high doses of all mAb except the IgG anti-FasL mAb G247-4, which is specific to a segment outside the FasL binding site. Interestingly, under identical conditions but in the presence of 2.4G2, G247-4 inhibited the cytotoxicity of FasL VP. In addition, G247-4 inhibited the FasL VP-mediated killing of FcR(-) Jurkat. The data demonstrate that FasL-expressing bioactive vesicles display a property heretofore unknown in bioactive agents that express FasL-mediated cytotoxicity. The mechanism of the Ab-mediated, FcR-dependent enhancement of cytotoxicity of bioactive vesicles and its physiological significance are discussed.     

Quantum dot-based fluorescence immunosorbent assay for the rapid detection of bacitracin zinc in feed samples

Bacitracin zinc (BAC), a polypeptide antibiotic, is utilized as a feed additive due to its ability to promote growth in animals. However, the abuse of BAC can lead to a great threat to food safety. Therefore, there is an urgent need to develop a rapid and sensitive detection method. In this study, a monoclonal antibody (mAb) against BAC with excellent sensitivity and specificity was obtained. For the first time, quantum dots (QDs) were conjugated with the prepared mAb against BAC and rabbit anti-mouse antibody to fabricate a direct and an indirect competitive fluorescence-linked immunosorbent assay (dc-FLISA and ic-FLISA) to detect BAC. The IC₅₀ of dc-FLISA and ic-FLISA were 0.28 ng/ml and 0.17 ng/ml, respectively. The limits of detection were 0.0016 ng/ml and 0.001 ng/ml, respectively, and the detection ranges were 0.0016–46.50 ng/ml and 0.001–35.65 ng/ml, respectively. In addition, the recovery rate of the two methods ranged from 93.5% to 112.0%, and the coefficient of variation (CV) was less than 10%. Therefore, the methods developed in this work have the merits of low cost, simple operation, and high sensitivity, which provide an effective analytical tool for BAC residue detection in feed samples.     

人生长分化因子15单克隆抗体制备、鉴定及药用价值初探

目的:制备稳定分泌抗人生长分化因子15(GDF15)单克隆抗体(m Ab)的杂交瘤细胞系,并对其分泌的m Ab进行鉴定。方法:根据人GDF15氨基酸序列特征,设计合成了8条能够免疫产生GDF15特异性抗体的抗原多肽,与VLP载体偶联后,免疫雌性BALB/c小鼠,利用杂交瘤技术制备鼠源抗人GDF15的m Ab,用间接ELISA检测m Ab腹水效价。结果:获得针对7个抗原多肽的12株稳定分泌抗人GDF15的杂交瘤细胞系,腹水m Ab效价可达1×104~1×109。结论:获得了针对不同抗原多肽的抗人GDF15的特异性m Ab,为进一步研发以GDF15为靶点的单克隆抗体抗肿瘤药物奠定了基础。     

天然产物莪术醇单克隆抗体的制备

为制备小分子化合物莪术醇的单克隆抗体,先将莪术醇(curcumol)与载体蛋白牛血清蛋白(BSA)偶联形成完全抗原,用基质辅助激光解吸飞行时间质谱法(MALDI-TOF-MS)鉴定莪术醇人工抗原的偶联率,然后采用杂交瘤技术获得杂交瘤株,并对其进行小鼠腹水的制备与纯化.结果表明:莪术醇半抗原与载体的偶联比为19.6,单克...     

Effect of epidermal growth factor on preimplantation development and its receptor expression in porcine embryos.

The present study aimed to determine the influence of exogenous epidermal growth factor (EGF) on in vitro preimplantation porcine embryo development and its mRNA expression for EGF receptor (EGFR). Oocytes were aspirated from abattoir ovaries, selected and cultured in defined, protein-free media for 44 hr before in vitro fertilization (IVF). Thirty-six hours after IVF, two-cell stage embryos were selected and treated or cultured until embryo treatment. In experiment 1, compact morulae were selected on day 4 after IVF and randomly allocated into 5 groups: NCSU 23 with PVA as group 1; NCSU 23 with PVA and 0.1 ng/ml, 1.0 ng/ml, 10.0 ng/ml EGF as group 2, 3, 4, respectively; NSCU 23 with 0.4% BSA as group 5. In experiment 2, treatment groups were the same as in experiment 1 except that 0.1% crystallized BSA was added to both washing media and all treatment groups instead of PVA. In experiments 3 and 4, two-cell stage embryos were treated and cultured in the same experimental design as experiments 1 and 2, respectively. RT-PCR was used to detect the mRNA expression of EGF receptor in compact morulae and blastocysts. The PCR products were subjected to direct DNA sequencing. There was no significant improvement in the development rate of embryos from compact morulae to blastocysts in the presence of various EGF concentrations (0.1, 1.0, 10.0 ng/ml) versus without EGF addition. They were all significantly lower than those embryos cultured in the continuous presence of 0.4% BSA. However, when a reduced concentration (0.1%) of crystallized BSA was added to all the treatment groups, a significantly lower rate of embryo development was observed in control media (NCSU23 with 0.1% crystallized BSA) compared with those developed in culture media with 0.4% BSA. With the addition of EGF at 10 ng/ml (with 0.1% BSA), embryo development rates were significantly improved over the control group (P < 0.05) and were as good as those rates in 0.4% BSA culture group. When embryos were selected and treated from the 2-cell stage, they did not develop to blastocyst stages after five more days' culture without any protein (BSA) or growth factor addition. When 0.1% BSA was included in the media, blastocyst formation rates were significantly improved by EGF addition at the concentration of both 1.0 or 10 ng/ml (P < 0.05) as compared to 0.0 or 0.1 ng/ml. EGFR mRNA was detected in both compact morulae and blastocyst stages of porcine embryos and confirmed by direct DNA sequencing. Our results indicate that IVM-IVF porcine embryo developmental rates could be improved by the addition of EGF in the culture media with the presence of a reduced amount of defined BSA (>97% albumin). However, EGF alone was not able to elicit any stimulatory effects on embryo development in the absence of protein supplementation. Further studies are needed to investigate the potential synergistic factors in embryo culture media to eventually define the porcine embryo culture media.     

Characterization of monoclonal antibodies identifying type and strain-specific epitopes of human immunodeficiency virus type 1

Summary Several hybridoma cell lines were raised against the highly cytopathic Zairian isolate of Human Immunodeficiency Virus (HIV), HIV1-NDK.The specificity of the secreted monoclonal antibodies (mAb) was demonstrated by immunoblotting, radioimmunoprecipitation and immunofluorescence. Two hybridoma cell lines secreted mAb reacting with independent epitopes of the NDK p17 capsid protein and its precursors. One, RL16.24.5, is specific for the NDK isolate whereas the other, RL16.45.1, along with anti-p25 RL16.30.1 mAb, bind all HIV1 isolates but not HIV2. Together with the previously described mAb RL4.72.1 those reagents define lentivirus subfamily (HIV1, HIV2, SIV) type/subtype (HIV1) and strain (HIVI-NDK) specific epitopes expressed on HIVl-NDK core proteins. The last mAb RL16.76.1 binds the env gene products gp160 and gp120.     

己烯雌酚特异性抗体的制备及鉴定

利用4-溴丁酸乙酯对小分子半抗原己烯雌酚(DES)进行活化,引入羧基活性基团,应用活泼酯法将其与牛血清白蛋白(BSA)偶联,合成DES-CP-BSA完全抗原,免疫新西兰长耳白兔,制备特异性抗体.结果显示:成功制备了DES完全抗原,且由此获得了特异性的DES抗体,效价达1.28×105,与己烷雌酚、双烯雌酚的交叉反应分别...