Western Blot Detection of Human Anti-Chikungunya Virus Antibody with Recombinant Envelope 2 Protein

Chikungunya virus (CHIKV), a tropical pathogen, has re-emerged and has massive outbreaks abruptly all over the world. Containing many dominant epitopes, the envelope E2 protein of CHIKV has been explored for the vaccination or diagnosis. In the present study, the antigenicity of a recombinant expressed intrinsically disorder domain (IUD) of E2 was tested for the detection of the antibody against CHIKV through western blot method. The gene of the IUD of E2 was inserted into 2 different vectors and expressed as recombinant GST-E2 and recombinant MBP-E2 fusion protein, respectively. Two kinds of fusion proteins were tested with 30 CHIKV patient sera and 30 normal sera, respectively. Both proteins were detected by 25 patients sera (83.3%) and 1 normal serum (3.3%). This test showed a relatively high sensitivity and very high specificity of the recombinant E2 proteins to be used as diagnostic antigens against CHIKV infection.     

Identification and localization of calcium-dependent protease II inNeurospora crassa andUromyces appendiculatus

Summary The existence of Ca²⁺-dependent protease II in crude extracts ofNeurospora crassa andUromyces appendiculatus was demonstrated by immunoblotting using specific antibodies. In both extracts two immunoreacting bands were observed. The molecular mass of the major band inN. crassa corresponded to 37 kDa, while that inU. appendiculatus was 43 kDa, similar to that previously reported forAllomyces arbuscula. Immunofluorescence of the enzyme was predominantly localized in the apical regions of germlings and growing hyphae, suggesting a functional role for the enzyme in hyphal growth.     

水稻矮缩病毒最外层外壳蛋白基因(S_2)cDNA克隆、序列分析及其在大肠杆菌中的表达

从水稻矮缩病毒（Ｒｉｃｅｄｗａｒｆｖｉｒｕｓ,ＲＤＶ）中国福建分离物中克隆分离了最外层外壳蛋白基因（Ｓ２）全长ｃＤＮＡ,并对其进行了序列分析,结果表明ＲＤＶＳ２ｃＤＮＡ全长３５１２ｂｐ,仅含一个３３４８ｂｐ的阅读框架,编码一个含有１１１６个氨基酸的蛋白（Ｐ２）。与基因库中已知基因序列比较,发现它与日本ＲＤＶＨ株系相应片段的核苷酸和氨基酸同源率分别为９４６％和９５４％,与轮状病毒ＶＰ２氨基酸序列有一定的同源性。Ｓ２核苷酸序列二级结构预测结果表明,５’端５０个核苷酸的二级结构为一个发夹结构和一个茎环结构。Ｐ２有４个富含亮氨酸的区域,位于Ｎ端亲水区域的１０个氨基酸（ＡＡ６９～７８）残基形成一个α螺旋,这些特点均与轮状病毒ＶＰ２的结构特征相似。ＳＤＳＰＡＧＥ和Ｗｅｓｔｅｒｎ印迹分析表明在大肠杆菌中分段高效表达了Ｓ２编码蛋白的Ｎ端和Ｃ端。     

Transgenic Rice Plants Produced by PEG-Mediated Plasmid Uptake into Protoplasts

Embryogenic cell suspension cultures were obtained from calli developed from mature rice seeds of a Japonica type Itahan cultivar, Roncarolo. Protoplasts were isolated and transformed by PEG-treated method with plasmid pHP23 carrying the NPT Ⅱ gene which encodes resistance to antibiotic G-418. Protoplast-derived colonies were selected in presence of the inhibitor. Plants were regenerated and transplanted into soil in the green house. The presence of foreign gene in the regenerated plants was verified by PCR and Southern analysis.     

粘虫核型多角体病毒凋亡抑制基因的定位,序列和启动子结构

以ＡｃＮＰＶ凋亡抑制基因ｐ３５为探针,与ＬｓＮＰＶＤＮＡ的限制性片段和ＬｓＮＰＶＤＮＡＥｃｏＲＶ片段杂交,发现ＥｃｏＲＶ５．５ｋｂ片段有强烈的杂交信号。将此片段亚克隆后,测定了１２４４ｂｐ序列,发现一个完整的ＯＲＦ,推导的３０２个氨基酸与ＡｃＮＰＶｐ３５蛋白有７０．４％的氨基酸同源性,证明所测ＯＲＦ为ＬｓＮＰＶ的ｐ３５基因。结构分析发现其５′端有早期基因启动子元件ＧＣ、ＡＣＧＴ和ＴＡＴＡｂｏｘ。有２２ｂｐ的顺向重复序列,包括由两个重叠的ＴＡＴＡｂｏｘ和上下游两个ＡＣＧＴｍｏｔｉｆ组成的两套启动子元件,这些结构特征与ＡｃＮＰＶ的凋亡抑制基因十分相似。     

橡胶延伸因子基因及3'端非编码区的克隆与序列分析

橡胶延伸因子ＲＥＦ（Ｒｕｂｂｅｒ Ｅｌｏｎｇａｔｉｏｎ Ｆａｃｔｏｒ）是橡胶生物合成中最重要的酶之一．作者利用ＲＥＦ基因序列设计并合成引物． 通过提取胶乳总ＲＮＡ 用ＲＴ法合成ｃＤＮＡ 后,通过ＰＣＲ技术扩增并克隆了ＲＥＦ基因和３端非编码区． 序列测定结果表明,ＲＥＦ基因全长４１４ 个碱基,编码１３８ 个氨基酸,３端非编码区长１１２ ｂｐ． 与Ｇｏｙｖａｅｒｔｓ Ｅ 等人发表的序列比较：ＲＥＦ基因同源率为１００％ ,３端非编码区有２ ｂｐ 的差异,同源率为９８％ ． 将所克隆的ＲＥＦ基因及３非编码区进行地高辛标记, 对胶乳和叶片总ＲＮＡ 进行点杂交分析,结果表明,胶乳总ＲＮＡ 呈阳性反应,叶片总ＲＮＡ 则呈阴性反应．     

Western blot免疫印迹法检测磷酸化蛋白表达条件优化研究

目的:探讨Western blot免疫印迹法不同转膜方法和不同抗原抗体比例对磷酸化蛋白表达的检测效果。方法:选择肌球蛋白轻链(myosin light chain,MLC)及其磷酸化蛋白作为研究对象,比较半干转印法、湿转法和1:3000、1:5000、1:10000等抗体稀释比例对磷酸化蛋白检测效果的影响。结果:半干转印法(恒压16V,30 min)观察到蛋白信号断续;而同样样品利用湿转法(恒压130 V,1h)检测发现信号连续且强度明显增高;对于磷酸化蛋白,半干转印法无法观察到磷酸化蛋白信号;而同样样品湿转法检测出现连续信号。统一利用湿转方法进行后续蛋白磷酸化检测,当抗体稀释比为1:3000时,结果出现非特异性条带;降低抗体稀释比为1:5000时无非特异性条带,且蛋白信号效果较好;抗体稀释比为1:10000时条带图像出现弥散且背景较高。结论:选择合适的转膜方式和抗原抗体比例有助于磷酸化蛋白表达检测。