用PCR法直接快速筛查重组阳性克隆

本研究目的是应用PCR法快速筛查插入有苯丙氨酸脱氨酶（PAL）cDNA重组阳性克隆,用于PCR扩增的引物是位于载体pET23b启动子处的T7启动子引物和位于目的基因PALc DNA3’端终止密码TAA处的引物,以灭菌吸头挑一单菌落加PCR体系扩增。结果：在筛查的3个克隆中,有2个阳性克隆,并且插入方向正确,经DNA序列测定得到进一步证实,结果表明,以PCR方法筛查重组阳性克隆,可以简便快速鉴定插入片段的大小和方向,不需提取质粒。     

利用PCR技术行反义寡核苷酸体外抗丁型肝炎病毒基因组RNA中核酶的研究

设计一对ＰＣＲ引物,其中上游引物的５’端除目的基因外,还加Ｔ７ＲＮＡ聚合酶启动子序列,以质粒（ｐＳＶＬＤ３）为模板,通过ＰＣＲ扩增出带有Ｔ７ＲＮＡ聚合酶启动子序列的１３９ｂｐ的ｃＤＮＡ片段,它含有丁型肝炎病毒（ＨＤＶ）基因组ＲＮＡ中核酶（Ｒｉｂｏｚｙｍｅ）区的ｃＤＮＡ该核酶具有自身裂解功能,经测序发现该ｃＤＮＡ有２个碱基变异,以此ＰＣＲ产物为模板,通过Ｔ７ＲＮＡ聚合酶,转录出核酶的前体,并观察到其     

抗人CD3单抗重,轻链可变区基因的体外扩增,克隆和序列分析

根据免疫球蛋白重链和轻链可变区基因５＇端序列和Ｊ区序列,化学合成适合于体外扩增抗体重、轻链可变区基因的二对引物。从体外培养的ＯＫＴ３杂交瘤细胞中提取总ＲＮＡ,反转录生成ｃＤＮＡ,以ｃＤＮＡ为模板,分别加入合成的重、轻链可变区引物进行ＰＣＲ,扩增出抗体重、轻链可变区基因片段。将扩增产物分别插入ｐＵＣ１９质粒,筛选出阳性克隆,用链终止法进行ＤＮＡ序列测定。所测重链可变区基因全长３５７ｂｐ,编码１１９个     

抗人CD3单抗重、轻链可变区基因的体外扩增、克隆和序列分析

根据免疫球蛋白重链和轻链可变区基因５’端序列和Ｊ区序列,化学合成适合于体外扩增抗体重、轻链可变区基因的二对引物。从体外培养的ＯＫＴ３杂交瘤细胞中提取总ＲＮＡ,反转录生成ｃＤＮＡ,以ｃＤＮＡ为模板,分别加入合成的重、轻链可变区引物进行ＰＣＲ,扩增出抗体重、轻链可变区基因片段。将扩增产物分别插入ｐＵＣ１９质粒,筛选出阳性克隆,用链终止法进行ＤＮＡ序列测定。所测重链可变区基因全长３５７ｂｐ,编码１１９个氨基酸,轻链可变区基因全长３２１ｂｐ,编码１０７个氨基酸。     

A组轮状病毒SA11VP6基因的克隆和表达

从ＳＡ１１ＶＰ６基因全序列克隆开始,设计一对两端带有酶切位点的引物,逆转录ＰＣＲ扩增出ＶＰ６全基因ＣＤＮＡ。经酶切后插入ＰＵＣ１９,构建了ＶＰ６全基因克隆ＰＲＡ６。再经酶切后插入痘苗病毒载休质凿ＰＪＳＡ１１７５中。利用Ｌｉｐｏｆｅｃｔｉｎ导入ＴＫ１４３细胞,利用ＴＫ基因和Ｌａｃ基因作为重组病毒的筛选标记。表达产物用单克隆抗体ＥＬＩＳＡ法检测,发现细胞培养上清和细胞裂解液都是阳性。Ｗｅｓｔｅｒｎ ｂ     

黄瓜花叶病毒香蕉株系的衣壳蛋白基因克隆和序列分析

对侵染香蕉的黄瓜花叶病毒广东３个株系的衣壳蛋白（ＣＰ）基因,进行了克隆和序列分析,以提纯病毒ＲＮＡ为模板,应用ＲＮＡ反转录酶（ＡＭＶ）合成ＣＰ基因ｃＤＮＡ再用ＴａｑＤＮＡ聚合酶进行ＰＣＲ扩增,通过常规基因克隆法扩增的ＣＰ基因克隆入载体,选取每一株系的ＣＰ基因与载体表面方向相同和相反的各一个克隆,进行插入片段的全序列分析,结果表明,每一重组克隆测序长约７５０个核苷酸,任一株系其插入方向相反的两个重组     

岸蟹（Carcinus maenas）金属硫蛋白cDNA及其基因的克隆

利用已知的Ｃ．ｍａｅｎａｓ金属硫蛋白氨基酸序列资料,用全简并的ＰＣＲ引物,从鳃组织总ＲＮＡ中扩增出两种金属硫蛋白ｃＤＮＡ片断,并将其克隆到ｐＧＥＭ－Ｔ载体中,序列测定表明,其中一种ｃＤＮＡ片断核苷酸序列和推知的Ｃ．ｍａｅｎａｓ金属硫蛋白核苷酸序列完全吻合;另一种ｃＤＮＡ片断则在３’端有较大变异。根据前者ｃＤＮＡ片段序列设计特异性引物,扩增并克隆了其编码区全长ｃＤＮＡ和其编码基因,测序结果表明,岸蟹     

水稻中编码甘油—3—磷酸转酰酶部分cDNA的克隆及序列分析

参照国外报道的几种双子叶植物的甘油－３－磷酸转酰酶的相对保守的氨基酸序列,设计并合成了一对简并引物,提取抗冷性强的水稻品种“丽梗２号”的ＲＮＡ,采用ＲＴ－ＰＣＲ技术,扩增出３１５ｂｐ的一般ｃＤＮＡ片段。扩增产物经纯化后直接克隆到ｐＧＥＭ－Ｔ载体系统中,经ＰＣＲ法鉴定,所得的重组质粒中含有３１５ｂｐ的片段。     

鼠I型可溶性白细胞介素1受体基因在昆虫细胞的克隆和表达

白细胞介素１（ＩＬ－１）是一种重要的细胞因子,具有广泛的生物学活性。它通过与细胞表面的白细胞介素１受体（ＩＬ－１Ｒ）结合而起作用,以杆状病毒为载体在昆虫细胞中克隆表达了小鼠Ｉ型可溶性白细胞介素１受体基因,以ＮＩＨ／３Ｔ３细胞ＲＮＡ为模板,采用ＲＴ－ＰＣＲ方法扩增得到小鼠ｓＩＬ－１ＲＩ的ｃＤＮＡ,克隆至杆状病毒转移载体ｐＡｃＧＰ６７Ｂ,将转移重组质粒与野生病毒ＡｃＮＰＶ ＤＮＡ共转染昆虫细胞Ｓｆ９,     

虹鳟鱼金属硫蛋白启动子的体外扩增、克隆及其序列分析

以含有虹鳟鱼金属硫蛋白基因的质粒ＤＮＡ为模板,以合成的两段３２个寡聚核苷酸为引物,经过ＰＣＲ扩增,合成了４３３ｂｐ的片段,把其克隆到ｐＢＬ－ＣＡＴ载体中,序列分析和限制酶切图谱表明所克隆的虹鳟鱼金属硫蛋白启动子含有４３３ｂｐ,含有ＴＡＴＡ和ＣＡＡＴ序列,与Ｈｏｎｇ报导的鳟鱼金属硫蛋白启动子的序列比较,其核苷酸泊同源率为１００％。     

Entwicklungsgeschichte und Ultrastruktur von Pollenkitt und Exine bei nahe verwandten entomophilen und anemophilen Angiospermensippen derAlismataceae,Liliaceae, Juncaceae,Cyperaceae, Poaceae undAraceae

Some closely related members of the monocotyledonous familiesAlismataceae, Liliaceae, Juncaceae, Cyperaceae, Poaceae andAraceae with variable modes of pollination (insect- and wind-pollination) were studied in relation to the ultrastructure of pollenkitt and exine (amount, consistency and distribution of pollenkitt on the surface of pollen grains). The character syndromes of pollen cementing in entomophilous, anemophilous and intermediate (ambophilous or amphiphilous) monocotyledons are the same in principal as in dicotyledons. Comparing present with former results one can summarize: 1) The pollenkitt is always produced in the same manner by the anther tapetum in all angiosperm sub-classes. 2) The variable stickiness of entomophilous and anemophilous pollen always depends on the particular distribution and consistency of the pollenkitt, but not its amount on the pollen surface. 3) The mostly dry and powdery pollen of anemophilous plants always contains a variable amount of inactive pollenkitt in its exine cavities. 4) A step-by step change of the pollen cementing syndrome can be observed from entomophily towards anemophily. 5) From the omnipresence of pollenkitt in all wind-pollinated angiosperms studied one can conclude that the ancestors of anemophilous angiosperms probably have been zoophilous (i.e. entomophilous) throughout.

     

Identification and Characterization of the First Equine Parainfluenza Virus 5

正Dear Editor,Parainfluenza virus 5 (PIV5), known as canine parainfluenza virus in the veterinary field, is a negative-sense,nonsegmented, single-stranded RNA virus belonging to the Paramyxoviridae family (Chen 2018). The virus was first reported in primary monkey kidney cells in 1954 (Hsiung1972), then it has been frequently discovered in various     

Pathogenic Characterization and Full Length Genome Sequence of a Reassortant Infectious Bursal Disease Virus Newly Isolated in Pakistan

<正>Dear Editor,Infectious bursal disease (IBD) is one of the most important diseases of the poultry. The IBD virus (IBDV), a nonenveloped virus belonging to the Birnaviridae family with a genome consisting of two segments of double-stranded RNA (segments A and B), targets B lymphocytes of bursa of Fabricious leading to immunosuppression. In Pakistan,poultry farming is the second biggest industry and IBD is the second biggest disease threating the poultry sector.However, there is limited genome information of IBDV     

Similar aerosol emission rates and viral loads in upper respiratory tracts for COVID-19 patients with Delta and Omicron variant infection

Highlights
1 Aerosol emission rates of Delta or Omicron patients were similar.
2 Viral loads in upper respiratory tract of Alpha, Delta and Omicron patients were similar.
3 Viral loads in upper respiratory tract of vaccinated or unvaccinated Delta patients had no difference.     

Recombinant human interferon-α1b inhibits SARS-CoV-2 better than interferon-α2b in vitro

Highlights
1) A comprehensive evaluation method for anti-SARS-CoV-2 drugs was established based on RT-qPCR, TCID₅₀ method, and immunofluorescence.
2) A significant antiviral effect of rHuIFN-α1b was shown with EC₅₀=0.12 IU/mL in Vero cells and EC₅₀=0.52 IU/mL in Calu-3 cells, which was better than rHuIFN-α2b (EC₅₀=0.25 IU/mL in Vero cells and EC₅₀=2.48 IU/mL in Calu-3 cells).
3) rHuIFN-α1b has a good potential in the application of anti-COVID-19 therapy.