牛生长激素cDNA的分子克隆

我们克隆了与牛生长激素Poly(A)⁺RNA互补的DNA(cDNA)。首先从小牛垂体中提纯总的Pply(A)⁺RNA,用AMV逆转录酶合成单链cDNA,以单链cDNA为模板合成双链cDNA,用多聚G及多聚c谱尾法将双链cDNA克隆到pBR322质粒的Pst I位点上,构建成牛垂体PoIy(A)⁺RNA的cDNA文库。以牛生长激素基因为探针,筛选出7个阳性菌落,经电泳鉴定有两个菌落(1号和2号)含有大于500bp的插入片段。1号克隆经酶切图谱、southeTn blot 杂交及序列分析证实含有牛生长激素的编码序列。     

野生大豆未成熟种子总mRNA的分离及其cDNA的分子克隆

用氯化锂沉淀法从野生大豆(G.soja)未成熟种子中制备总RNA,经oligo(dT)-纤维素柱亲和层析,获得总mRNA,在兔网织红细胞体系中表现出一定翻译活性。以总mRNA为模板,oligo(dT)_(12)(?)为引物,反转录酶催化合成第一链cDNA,RNase H-DNA聚合酶Ⅰ协同合成第二链cDNA。双链cDNA的长度大约为200—5000 bp,且不存在发夹结构。将双链cDNA修补后钝端连接到pUC 19质粒的Sma 1位点,转化E.coli JM107,获得800多个白色重组子克隆。快速电泳检测及酶切分析表明,多数重组子带有插入片段,其中3个重组子的插入片段长度大致为1700 bp、2600 bp和1400 bp。     

柔嫩艾美耳球虫孢子化卵囊cDNA文库的构建

用建立表达性文库的方法 ,构建了Eimeriatenella孢子化卵囊噬菌体ZAP表达性cDNA文库。首先用TRIzol试剂盒从E .tenella孢子化卵囊中提取总RNA ,再用Oligo(dT)₁₂纤维素柱从总RNA中分离mRNA ,以mRNA为模板 ,Oligo(dT)₁₈Linker Primer为引物 ,反转录合成cDNA第一链 ,再在DNA聚合酶Ⅰ作用下置换合成第二链cDNA。cDNA第二链合成后用PfuDNA聚合酶补平XhoⅠ位点 ,再与EcoRⅠAdapters连接 ,经XhoⅠ酶切后 ,凝胶电泳回收 500bp～4.0kp之间的cDNA片段 ,纯化后的双链cDNA与载体ZAPExpressvector连接。体外包装后得到E .tenella孢子化卵囊的cDNA表达性文库 ,经测定该文库的容量为 6×10⁶ ,扩增后文库的滴度为 1×10¹¹ Pfu mL ,经PCR测定 ,该文库的重组率为 96%。     

北京鸭珠蛋白mRNA的分离纯化及其cDNA的合成

 从人工贫血的北京鸭网织红细胞中直接提取总RNA,经Oligo(dT)-纤维素柱层析分离获得珠蛋白mRNA,并经蔗糖密度梯度离心首次得到了电泳单一条带的北京鸭球蛋白mRNA。从凝胶电泳以及蔗糖密度梯度离心鉴定其沉降系数为9S。在麦胚无细胞体外翻译体系中测定了它们的蛋白翻译活力。鸭珠蛋白mRNA促进了~3H-亮氨酸参入新生蛋白的活力,达到对照组的10倍。所翻译的蛋白产物在SDS-聚丙烯酰胺凝胶上的电泳行为与天然鸭珠蛋白一致。 经Oligo(dT)-纤维素及蔗糖密度梯度离心提纯的珠蛋白mRNA,在AMV反转录酶及DNA聚合酶的作用下,分别合成了单链及双链cDNA。其双链链长,经凝胶电泳分析,约为500碱基对。     

虎纹捕鸟蛛毒腺cDNA文库的构建

从 2 5只虎纹捕鸟蛛 (Selenocosmia huwena)中得到约 0 .6g左右的毒腺 ,从中提取出5μg m RNA.以此 m RNA为模板 ,经反转录合成双链 c DNA.再经包装成 c DNA文库 .文库的滴度达到 3.2× 1 0 7pfu/m L     

苜蓿豆血红蛋白互补脱氧核糖核酸(cDNA)的合成及其无性繁殖

本文介绍了 cDNA 合成的详细方法。用苜蓿豆血红蛋白 poly A( )mRNA 作模板,在反转录酶作用下合成单股 cDNA(ss-cDNA),然后在 E.coli DNA 聚合酶 I 作用下合成双股 cDNA(ds-cDNA)。采用同聚物尾接方法和合成的内切酶寡核苷酸衔接物连接方法,将 ds-cDNA 与质粒 pBR322 DNA 重组,进行转化,得到对氨苄青霉素抗性(Amp~r)和四环素敏感(TeL~s)的75个转化子。经 cDNA-mRNA 的杂交选择和无细胞体外转译的初筛结果,有两个转化子具有与苜蓿豆血红蛋白 mRNA 有同源性的 cDNA。这种 cDNA 的性质有待于进一步研究.     

银染RD-PCR方法分离基因片段

从正常培养的SH-SY5Y细胞中提取总RNA,经oligo(dT)纤维素柱纯化分离出mRNA,然后以oligo(dT18)为锚定引物反转录生成单链cDNA再以此为模板合成DNA的第二条链;将双链DNA经Sau3AI酶切之后,接上接头,经通用引物和选择性引物进行扩增;采用5%非变性PAGE胶电泳分离后,用银染的方法显示DNA条带;在直视下回收DNA带,经过扩增后克隆入pMD18-T载体中并鉴定。结果建立了非放射性同位素的银染RD-PCR方法,用于分离基因片段。     

野生大豆种子cDNA文库的构建与分析

为了分离与鉴定野生大豆优良基因,以双高型优质野生大豆的近成熟种子为材料,采用裂解法提取了总RNA;以Oligo（dT）为引物,经SA—PMPS法分离出mRNA,反转录酶催化合成cDNA,并以cDNA第一链为模板在DNA聚合酶Ⅰ的作用下合成cDNA第二链,双链cDNA经加接头等步骤,成功构建了野生大豆cDNA文库。文库的重组率约为93．7％,PCR检测重组克隆的插入片段平均大于1000bp,测序片断大于500bp,表明构建的近成熟种子cDNA文库质量较高,为进一步进行EST测序和全长克隆打下了基础。     

黄地老虎颗粒体病毒cDNA文库的构建

以感染黄地老虎颗粒体病毒（Agrotis segetum ganulosis virus,AsGV)黄地老虎幼虫为材料提取总RNA,分离mRNA,并反转录合成cDNA ,构建了包括黄地老虎（Agrotis segetum,As)幼虫和黄地老虎颗粒体病毒的cDNA 文库。用Eco RI和HindⅢ限制性内切酶酶切AsGV基因组DNA,制备地高辛探针,与上述总RNA,mRNA,cDNA 杂交,从文库中筛选出AsGV的阳性克隆1081个,经cDNA测序,cDNA 编码序列与基因组编码序列相符。并根据基因组阅读框序列合成引物,PCR扩增出59个阅读框的编码基因,也完全与基因组序列相符。     

猪生长激素cDNA的分子克隆

用改进的氯化锂沉淀沉法和寡聚(dT)一纤维素亲和层析法由猪垂体制得总mRNA。以此总mRNA为模板,合成cDNA。钝端连接重组到质粒pUC19的SmaI位点,转化E.coli JM107,筛选出阳性克隆。用限制性内切酶酶切签定及5’端部分核苷酸序列分析证明:克隆了全长猪生长激素cDNA,其长度约为896bp。     

Carp growth hormone: molecular cloning and sequencing of cDNA

cDNA clones of the fish Cyprinus carpio growth hormone (GH) mRNA have been isolated from a cDNA library prepared from carp pituitary gland poly(A)+RNA. The nucleotide sequence of one of the carp GH cDNA clones containing an insert of 1164 nucleotides (nt) was determined. The cDNA sequence was found to encode a polypeptide of 210 amino acids (aa) including a signal peptide of 22 aa and to contain 5' and 3' untranslated regions of the mRNA of 36 and 498 nt, respectively. The carp GH presents a 63% amino acid sequence homology with the salmon GH, has structural features common with other GH polypeptides of mammalian or avian origin and contains domains of conserved sequence near the N- and C-terminal regions. Southern blot hybridization of carp genomic DNA with GH cDNA probes shows the presence of at least two GH-coding sequences in the fish genome.     

Cloning of chicken lysozyme structural gene sequences synthesized in vitro.

Double-stranded chicken lysozyme cDNA was synthesized from an oviduct mRNA fraction enriched for lysozyme mRNA. The ds-cDNA was inserted into the BamHI site of plasmid pBR322 using chemically synthesized DNA linker molecules containing the BamHI restriction endonuclease cleavage site. After bacterial transformation, colonies carrying lysozyme DNA were identified by hybridization with highly purified lysozyme cDNA. The 555 base pairs long cloned DNA fragment of one recombinant plasmid was isolated and characterized by restriction endonuclease digestion. The DNA sequence of selected parts of the inserted DNA is as predicted from the amino acid sequence of prelysozyme. The sequence data allows the unambiguous location of the coding region within lysozyme mRNA.     

鲤鱼(Cyprinus carpio)生长激素基因克隆及原核表达

采用逆转录—聚合酶链式反应（ＲＴ－ＰＣＲ）方法,从鲤鱼脑垂体总ＲＮＡ中扩增出编码鲤鱼生长激素（ＧＨ）成熟肽基因序列．定向克隆至质粒ｐＵＣ１８,克隆的鲤鱼ＧＨｃＤＮＡ不含信号肽序列并以新的起始密码子ＡＴＧ取代鲤鱼ＧＨｃＤＮＡ第１个密码子ＴＣＡ．序列分析表明,与Ｋｏｒｅｎ报道的鲤鱼ＧＨｃＤＮＡ相比有两个碱基差异,但推断的氨基酸序列完全一致．将鲤鱼ＧＨｃＤＮＡ定向克隆至原核表达载体ｐＢＶ２２０,构建成重组鲤鱼ＧＨ基因表达载体ｐＢＶｃＧＨ８．ＳＤＳ－ＰＡＧＥ和薄层扫描分析表明：经４２℃诱导,ｐＢＶｃＧＨ８在大肠杆菌中可表达一分子量约２２０００的特异蛋白,表达量占细胞总蛋白的２９．２％．该基因重组的鲤鱼ＧＨ添加到饲料中投喂罗非鱼,证实有明显的促进生长作用     

Rapid synthesis and cloning of complementary DNA from any RNA molecule into plasmid and phage M13 vectors

We describe several modifications of the Gubler and Hoffman procedure [Gene 25 (1983) 263-269] for complementary DNA (cDNA) synthesis that expand the versatility of this method for the rapid synthesis and cloning of double-stranded (ds) cDNA. These modifications include: (1) The combination of first and second strand synthesis into a single two-step reaction, which reduces the time for synthesis of blunt-ended ds-cDNA to less than 4 h. (2) The use of random hexadeoxyribonucleotide primers (RP) for the synthesis of ds-cDNA, which allows the synthesis of cDNA from any RNA template. (3) The combined use of random primers and DNA ligase treatment of cDNA/RNA hybrids prior to second-strand synthesis, which promotes the production of nearly full length ds-cDNA molecules. (4) The use of gel filtration to size-fractionate ds-cDNA, which allows the selection of specific size classes of ds-cDNA for cloning. (5) The use of blunt-end ligation to insert the ds-cDNA into the vector, which reduces the total time required for the construction of cDNA libraries to less than 24 h.     

Human apolipoprotein A-II: complete nucleic acid sequence of preproapoA-II

The complete cDNA nucleic acid sequence of preproapolipoprotein (apo) A-II, a major protein constituent of high density lipoproteins, has been determined on clones from a human liver ds-cDNA library. Clones containing ds-cDNA for apoA-II were identified in the human liver ds-cDNA library using synthetic oligonucleotides as probes. Of 3200 clones screened, 4 reacted with the oligonucleotide probes. The DNA sequence coding for amino acids ?17 to +17 of apoA-II were determined by Maxam-Gilbert sequence analysis of restriction fragments isolated from one of these clones, pMDB2049. The remainder of the cDNA sequence was established by sequence analysis of a primer extension product synthesized utilizing a restriction fragment near the 5'-end of clone pMDB2049 as primer with total liver mRNA. The apoA-II mRNA encodes for a 100 amino acid protein, preproapoA-II that has an 18 amino acid prepeptide and a 5 amino acid propeptide terminating with a basic dipeptide (Arg-Arg) at the cleavage site to mature apoA-II.