富钙灵芝的深层培养

目的:研究灵芝深层发酵过程中钙对灵芝生长及钙的富集作用的影响。方法:用重氮化法将ABA分别与牛血清白蛋白(BSA)、卵清白蛋白(OVA)连结,得到ABA的免疫抗原和包被抗原,并采用紫外全波长扫描和SDS-PAGE鉴定合成的抗原;以经过鉴定的抗原免疫白兔,制备出ABA的多克隆抗体;采用间接酶联免疫法对抗血清进行效价检测;通过离子交换层析法获得纯化的抗体。结果:当培养基中硝酸钙的添加量达到600mg/100g时,灵芝生长和富集钙的效果最好;添加量超过800mg/100g时,灵芝富集钙的效果明显降低。富钙灵芝中有机钙的含量约占总钙量的97.30%。结论:在培养基中添加低浓度的钙,对灵芝的生长和钙的富集有促进作用,为开发新型有机钙提供了依据。     

除草剂2,4-D特异性多克隆抗体的制备及鉴定

目的是制备特异性的2,4-D多克隆抗体.用活性酯和混合酸酐法将半抗原2,4-D分别与牛血清白蛋白(BSA)和卵清蛋白(OVA)共价偶联;以2,4-D-BSA作免疫原免疫两只新西兰白兔;以2,4-D-OVA包被96孔酶标板,用间接酶联免疫吸附分析(ELISA)法检测抗血清效价和竞争性ELISA法检测2,4-D.结果显示2,4-D与BSA和OVA的偶联比率分别为32∶1和18∶1,抗血清效价＞6.4×105,在优化条件下2,4-D的最低检测限达37ng/mL.实验成功地制备了2,4-D特异性多克隆抗体,为其ELISA方法的建立提供了条件.     

圆弧青霉菌毒素-青霉酸人工抗原的合成与鉴定

为建立圆弧青霉毒素-青霉酸的免疫学检测方法, 研究了青霉酸(PA)的人工抗原合成.通过碳二亚胺法将青霉酸(PA)分别与牛血清白蛋白(BSA)和卵清蛋白(OVA)联结, 得到青霉酸人工抗原PA-BSA和PA-OVA.采用紫外扫描光谱法、SDS-PAGE和动物免疫试验对合成的抗原进行鉴定.结果显示联结后的人工抗原特征性吸收峰出现偏移, PA与BSA的偶联比为23.2:1, PA与OVA的偶联比为10.4:1.以PA-BSA为免疫抗原免疫小鼠, PA-OVA为包被抗原, 采用间接ELISA检测抗血清, 其效价达到1:12 800.表明青霉酸的人工抗原已合成, 为建立有效的免疫检测方法提供了基础.     

圆弧青霉菌毒素-青霉酸人工抗原的合成与鉴定

为建立圆弧青霉毒素-青霉酸的免疫学检测方法, 研究了青霉酸(PA)的人工抗原合成。通过碳二亚胺法将青霉酸(PA)分别与牛血清白蛋白(BSA)和卵清蛋白(OVA)联结, 得到青霉酸人工抗原PA-BSA和PA-OVA。采用紫外扫描光谱法、SDS-PAGE和动物免疫试验对合成的抗原进行鉴定。结果显示联结后的人工抗原特征性吸收峰出现偏移, PA与BSA的偶联比为23.2:1, PA与OVA的偶联比为10.4:1。以PA-BSA为免疫抗原免疫小鼠, PA-OVA为包被抗原, 采用间接ELISA检测抗血清, 其效价达到1:12 800。表明青霉酸的人工抗原已合成, 为建立有效的免疫检测方法提供了基础。     

猪圆环病毒2型Cap蛋白多克隆抗体的制备及鉴定

旨在制备猪圆环病毒2型(PCV2)Cap蛋白的多克隆抗体。以PCV2毒株(CAU0673)DNA为模板进行PCR,扩增目的片段大小约为702 bp,构建pET30a-PCV2-Cap重组质粒,转入大肠杆菌BL21(DE3),IPTG诱导表达;对目的蛋白进行NiNTA树脂亲和层析纯化、复性,并进行SDS-PAGE和Western blot鉴定;将纯化后的重组Cap蛋白与弗氏佐剂混匀乳化,经背部皮下多点注射4次,免疫新西兰大耳白兔,制备成兔抗Cap蛋白多克隆抗体,采用Western blot和间接免疫荧光试验(IFA)验证兔抗血清特异性,并用间接ELISA测定抗血清抗体效价。PCR、双酶切和测序鉴定结果表明,重组质粒pET30a-PCV2-Cap构建正确;重组Cap蛋白以包涵体的形式表达,大小约为34 kD,复性后重组Cap蛋白可与PCV2阳性猪血清发生特异性反应;制备的多克隆抗体与PCV2重组Cap蛋白和全病毒抗原均可发生反应,ELISA抗体效价 1∶12 800,显著高于商品化疫苗组。     

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。     

2种石房蛤毒素完全抗原交联方法的比较及多克隆抗体的制备

[目的]探究石房蛤毒素(STX)完全抗原制备方法和STX多克隆抗体免疫方案。[方法]通过碳二亚胺法(EDC)和高碘酸盐法(periodate reaction)2种交联方法,将小分子石房蛤毒素与牛血清白蛋白(BSA)、鸡卵清蛋白(OVA)和孔血蓝蛋白(KLH)分别进行交联,制备了6种形式STX完全抗原,并对交联物进行琼脂糖凝胶电泳鉴定和紫外吸收峰迁移变化鉴定。分别将EDC法和高碘酸盐法交联的STX-BSA、STX-KLH 4种完全抗原作为免疫原,对Balb/c小鼠进行免疫,获得STX多克隆抗体。通过间接ELISA法,对不同方法制备的多克隆抗体进行分析比较。[结果]在石房蛤毒素完全抗原的制备中,在交联方法的选择上,EDC法较高碘酸盐法更具优势;而在免疫原的选择上,STX-BSA完全抗原效果最好。[结论]本研究探究了2种制备STX完全抗原的方法,为今后多克隆抗体生产以及特异性单克隆抗体筛选提供数据支撑。     

钼还原菌中TrxR的原核表达及多克隆抗体制备

[目的]构建枯草芽孢杆菌硫氧还蛋白还原酶(thioredoxin reductase,TrxR)原核载体,表达、纯化TrxR重组蛋白,制备并鉴定多克隆抗体。[方法]通过分子克隆获得TrxR蛋白的表达菌株;利用镍离子亲和层析获得纯化的TrxR重组蛋白,免疫兔子制备TrxR蛋白多克隆抗体;采用ELISA法测定抗体效价;Western Blot检测抗血清的特异性。[结果]TrxR重组载体双酶切结果与DNA测序鉴定结果一致,蛋白表达纯化条带大小与预测一致。ELISA法测定抗血清效价为7×104,Western Blot证实抗血清有较高的特异性。[结论]成功克隆、表达与纯化TrxR重组蛋白,制备并鉴定兔子多克隆抗体,为TrxR的生物学功能研究奠定基础。     

棉蚜His-CYP6J1融合蛋白的分离纯化及多克隆抗体的制备

通过亲和层析法纯化棉蚜His-CYP6J1融合蛋白,制备及鉴定其多克隆抗体。采用镍离子金属螯合亲和层析柱分离纯化棉蚜His-CYP6J1融合蛋白,SDS-PAGE检测纯化产物。用纯化得到的His-CYP6J1融合蛋白免疫小鼠,制备抗棉蚜P450CYP6J1多克隆抗体;采用ELISA法检测抗体效价;免疫组化法检测抗血清特异性。结果表明,纯化的His-CYP6J1融合蛋白免疫小鼠后得到多抗血清,ELISA法检测抗血清效价达1∶200 000。免疫组化结果显示,此多克隆抗体能够与棉蚜P450 CYP6J1天然蛋白特异性结合,却在棉铃虫没有发现相应的结合反应。上述结果对研究棉蚜单一P450蛋白结构、功能及其在棉蚜抗药性形成过程的作用奠定基础。     

猪δ冠状病毒(PDCoV)N蛋白的原核表达及多克隆抗体制备

旨在表达和纯化猪δ冠状病毒(PDCoV)N蛋白并制备该蛋白的多克隆抗体。以RT-PCR扩增PDCoV N基因并与表达载体pET-28a构建重组质粒,转化Transetta(DE3)菌株诱导表达,SDS-PAGE鉴定融合蛋白表达,以纯化的N蛋白免疫家兔制备多克隆抗体,Western blot验证兔抗血清特异性,间接ELISA测定抗血清效价。利用间接免疫荧光试验(IFA)、免疫荧光试验(IF)、流式细胞术(FCM)鉴定其诊断应用价值。重组N蛋白为可溶性表达,大小约为44 kD,制备的兔抗N蛋白抗体效价可达1∶204 800。IFA与FCM试验证实该抗体能与PDCoV特异性结合,与PEDV及TGEV无交叉反应,IF试验表明该抗体可用于检测小肠组织中的PDCoV。     

Characterization and use in ELISA of a new monoclonal antibody for quantitation of abscisic acid in senescing rice leaves

A new monoclonal antibody (mAb) was generated against abscisic acid (ABA), and an indirect enzyme-linked immunosorbent assay (ELISA) using this mAb was developed for convenient quantitative analysis of ABA levels in rice leaf extracts. The mAb, raised against (+-)-ABA conjugated to bovine serum albumin (BSA) through its carboxyl group (C₁), reacted preferentially with the (+)-ABA enantiomer, and equally well with both free and methyl-ester (+-)-ABA. Cross-reactivity with several ABA-related compounds was negligible. Linearity was obtained between 3 and 1000 pmo1 of (+)-ABA. The ABA-mAb was further used to quantitate pmol quantities of (+)-ABA in attached and detached rice leaves. Results obtained with such ELISA quantitation showed an increase in the free ABA content of detached rice leaves at progressive stages of senescence, which was regarded as a senescence-related response. This quantitation compared favorably with other presently used techniques for ABA determination, with regard to their detection limits, cost and assay time. The results suggest that the combination of a specific mAb with a sensitive ELISA technique is quite promising for quantitation of ABA.     

Changes of free and conjugated abscisic acid and phaseic acid in xylem sap of drought-stressed sunflower plants

Abscisic acid (ABA), conjugated abscisic acid, phaseic acid (PA), and conjugated phaseic acid were determined by enzyme-linked immunosorbent assay (ELISA) and gas chromatography (GC) in xylem sap of well-watered and drought-stressed sunflower plants. Conjugated ABA and conjugated PA were determined indirectly after chemical or enzymatic hydrolysis. Conjugated ABA was found to be the predominant ABA metabolite in xylem sap. In xylem sap from well-watered plants at least five, and in sap from drought-stressed plants at least six alkaline hydrolysable ABA conjugates were found. One of them corresponds chromatographically (HPLC) with abscisic acid glucose ester (ABAGE). Under drought conditions the concentrations of ABA, alkaline hydrolysable ABA conjugates, -glucosidase hydrolysable ABA conjugates, PA, and conjugated PA increased. After rewatering the drought-stressed plants, the ABA and the conjugated ABA content decreased. The possible function of the ABA conjugates in the xylem sap as a source of free ABA is discussed.     

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...     

Estimation of endogenous contents of phytohormones during internode development in <Emphasis Type="Italic">Merremia emarginata</Emphasis>

During the entire period of internode growth of Merremia emarginata contents of gibberellic acid (GA₃), phenyl-acetic acid (PAA), indole-3-acetic acid (IAA, free and conjugated) and abscisic acid (ABA, free and conjugated) were estimated by ELISA using polyclonal antibodies raised against each hormones. At the time of internode elongation free auxin content was low and increased with the decrease in the rate of elongation. In contrast, conjugated IAA showed declining trend where free IAA content was remarkably high, suggesting thereby that conjugated IAA might have mobilized during the later phase of internode development. The endogenous GA₃ contents were high as compared to other hormones; however, no significant role of GA₃ was discernible in elongation growth. Conjugated ABA contents remained very low during the elongation growth and increased thereafter.     

A monoclonal antibody for the detection of conjugated forms of abscisic acid in plant tissues

A mouse monoclonal antibody against abscisic acid (ABA) was produced and characterized. It was raised using ABA conjugated to the carrier protein through the carboxyl (Cl) group as immunogen. It did not discriminate between free ABA or its ester derivatives. This antibody, which is the first monoclonal against Cl-conjugated ABA, shows interesting characteristics. It has high affinity (K_a=1.5 × 10⁹ L/mol) and specificity. Compounds structurally similar to ABA, such as phaseic acid, dihydrophaseic acid, and both the 2,trans-isomer and the (R)-enantiomer of ABA, are not reactive. The narrow linear range of the standard curve (0.018–1.8 pmol) ensures great precision of the assay. This monoclonal antibody has been used for the quantification of ABA conjugates in crude aqueous extracts of bean leaves by radioimmuno-assay (RIA). The fractionation of the extracts by high-performance liquid chromatography (HPLC) confirmed the absence of cross-reacting compounds. Because of its affinity and specificity, in combination with antibodies against free ABA, this antibody should be a sound tool for studying the metabolism and immunolocalization of ABA in plant tissues.     

Immunolocalization of abscisic acid by monoclonal antibodies in Lavandula stoechas L. leaves

A monoclonal antibody was used to localize abscisic acid in Lavandula stoechas L. plants treated with 1 M abscisic acid. ABA localization was performed using EDC as the only fixative, which preserves antigenicity and improves the specificity of monoclonal antibodies. A relationship was observed between the effect of abscisic acid on chloroplast ultrastructure and ABA accumulation. Gold particles accumulated on swollen chloroplasts. Our findings provide cytochemical evidence that the chloroplast is a trapping compartment, and yield valuable information on the relation between chloroplast ultrastructure and ABA accumulation.Abbreviations ABA abscisic acid - BSA bovine serum albumin - EDC 1-(3-dimethyl-aminopropyl)-3 ethyl carbodiimide - IgG immunoglobulin G - mAB monoclonal antibodies - pAB polyclonal antibodies