人肝再生增强因子阅读框的cDNA克隆和序列分析

目的获取人肝再生增强因子（ＡＬＲ）阅读框的ｃＤＮＡ克隆，为进一步研究打下基础。方法从人胎肝中提取总ＲＮＡ作为模板，以寡聚ｄＴ为引物逆转录台成第一链ｃＤＮＡ，用我们设计的引物和ＰＣＲ方法扩增双链ｃＤＮＡ。ＰＣＲ产物约３８０ｂｐ并被亚克隆人ｐＵＣ１９，经测序和ＰＣＧＥＮＥ软件分析。结果获得人ＡＬＲ完整阅读框的ｃＤＮＡ片段，长度为３７８ｂＰ。与大鼠ＡＬＲ和最近报道的人ＡＬＲ序列比较同源性分别为８６、５％和９９．２％。结论成功地克隆人ＡＬＲ完整阅读框的ｃＤＮＡ，同时提示人ＡＬＲ参与了人胎儿晚期发育过程中胎肝的生长调节。     

含CD44 cDNA片段细胞系的构建及在肝癌中的应用

目的构建含ＣＤ４４ｃＤＮＡ质粒ＰＧＥＸ２Ｔ和ＰＡＺ的单克隆细胞系．并利用此细胞系合成特异的ＣＤ４４ｃＤＮＡ探针，原位检测肝癌中ＣＤ４４ｍＲＮＡ的表达．方法应用常规的质粒转化方法将质粒ＰＧＥＸ２Ｔ和ＰＡＺ转入ＤＨ５菌株中得到单克隆细胞系．并以从此细胞系中提取的质粒为模板，应用ＰＣＲ法合成ＣＤ４４ｃＤＮＡ探针．应用原位杂交法检测肝细胞癌中ＣＤ４４ｖ６ｍＲＮＡ的表达．结果从ＤＨ５１菌株中提取的质粒含ＣＤ４４（２ｖ－１０ｖ）ｃＤＮＡ（１１ｋｂ），从ＤＨ５２菌株中提取的质粒含全长的ＣＤ４４ｃＤＮＡ（１８ｋｂ），ＰＣＲ合成的ＣＤ４４ｖ６ｃＤＮＡ探针长１４０ｂｐ．ＣＤ４４ｖ６ｍＲＮＡ的阳性检出率：转移高危组为８００％（８／１０），转移低危组为２１７％（５／２３），两者相差非常显著（Ｐ＜００１）．结论从单克隆细胞系ＤＨ５１和ＤＨ５２中提取的质粒，经ＰＣＲ扩增可得到特异的ＣＤ４４ｃＤＮＡ探针．肝细胞癌中ＣＤ４４ｖ６ｍＲＮＡ的表达与肿瘤的转移倾向呈正相关．     

小鼠肝再生增强因子cDNA的克隆化与序列分析

目的克隆小鼠肝再生增强因子（ＡＬＲ）的ｃＤＮＡ，并对其同源性序列进行检索与分析。方法以 人和大鼠的ＡＬＲ ｃＤＮＡ作为参考，以ＢＬＡＳＴ为检索途径，对美国国立生物工程学信息中心（ＮＣＢＩ）建立的核苷酸 序列数据库（ＧｅｎＢａｎｋ）进行检索，检出了小鼠ＡＬＲ基因组ＤＮＡ，ＧｅｎＢａｎｋ收录号为Ｕ４０４９４。根据大鼠、人ＡＬＲ 与小鼠ＡＬＲ同源性原则，设计了小鼠ＡＬＲ的特异性引物，以聚合酶链反应（ＰＣＲ）技术扩增获得了小鼠ＡＬＲ ｃＤＮＡ 片段，测序并进行核苷酸序列以及编码产物的氨基酸残基序列的同源性分析。结果获得了小鼠ＡＬＲ ｃＤＮＡ基 因片段，全长为５５９个核苷酸（ｎｔ），编码区为３７５ｎｔ，编码由１２５个氨基酸残基组成的多肽，计算分子量为１．５ × １０~４， 与人、大鼠及酵母ＥＲＶ１基因高度同源。结论小鼠ＡＬＲ ｃＤＮＡ编码一种酵母ＥＲＶ１的同源蛋白质。     

转染血管紧张素Ⅱ受体（AT1）反义核苷酸对血管平滑肌细胞增殖的 …

目的：评价转染ＡＴ１我核菩酸（ＡＴ１Ａ）对血管平滑肌细胞（ＶＳＭＣｓ）血管紧张Ⅱ（ＡｎｇⅡ）受体亚型ｍＲＮＡ表达、蛋白激酶Ｃ（ＰＫＣ）、丝裂素活化蛋白激酶Ｐ＾３８（Ｐ＾３８ＭＡＰＫ）蛋白表达,及蛋白核酸合成的作用。方法：ＲＴ－ＰＣＲ克隆ＡＴ１ ｃＤＮＡ序列（４７６ｂｐ）,将克隆的ＡＴ１ ｃＤＮＡ反向插入ＰｃＤＮＡ３．１,构建一完整的含ＡＴ１Ａ的质粒（ＰＡＴ１Ａ）,测序鉴定。转染培养的大鼠ＶＳＭＣｓ     

人淋巴细胞免疫反应性生长激素 cDNA 的克隆、序列测定与垂体生长激素 cDNA 的比较

从激活的人Ｔ淋巴细胞λｇｔ１０ｃＤＮＡ文库中筛选含免疫反应性生长激素ｃＤＮＡ序列的克隆，并分析其核苷酸序列。结果表明人淋巴细胞免疫反应性生长激素全部阅读框架的ｃＤＮＡ序列与人垂体生长激素的完全相同。在基因水平证实了神经内分泌与免疫系统共用信息传递因子的理论。     

初步筛选心血管相关基因——表达序列片段测定法

目的从正常人胎儿心脏ｃＤＮＡ文库中，用ＤＮＡ自动测序的方法筛选心血管相关基因。方法（１）人胚胎心ｃＤＮＡ文库的构建；（２）挑取有目的基因片段插入的噬菌斑，选用正向Ｔ３和反向Ｔ７引物做ＰＣＲ扩增；（３）直接使用其ＰＣＲ产物做ＤＮＡ测序模板，用荧光标记的Ｔ３Ｂ引物做再次ＰＣＲ扩增；（４）用ＤＮＡ测序仪（ｐｈａｒｍａｃｉａ）测定基因表达序列片段（ＥＳＴｓ）；（５）将所获得的ＥＳＴｓ输入Ｇｅｎｅｂａｎｋ数据库做同源序列分析。结果（１）所构建的人胎儿心脏ｃＤＮＡ文库的库容量为１×１０９克隆，平均目的基因片段１．２～１．５ｋｂ，ＰＣＲ产物阳性克隆检出率高达７０％以上，用６％尿素－聚丙烯酰胺凝胶电泳５ｈ，可测ｃＤＮＡ序列片段２１０～５５０ｂｐ，平均３００ｂｐ；（２）在所测３１３２个克隆的ＥＳＴｓ中，新ＥＳＴｓ为４７．４％，已知序列片段为４４．１％。结论利用人胎儿心脏ｃＤＮＡ文库测定ＥＳＴｓ是筛选心血管基因的有效方法；构建高质量的ｃＤＮＡ文库是成功测定ＥＳＴｓ的保证。     

肝再生增强因子的作用机制

人和动物血清中存在着促进肝再生的蛋白质因子 ,早期的研究中统称为肝刺激物质 (ＨＳＳ) [1,2 ] 。研究表明 ,肝再生增强因子 (ＡＬＲ)是ＨＳＳ的主要成分[1] 。有关ＨＳＳ主要功能性成分及其作用机制的研究 ,不仅有助于了解肝脏再生的生物学机制 ,而且有助于新型治疗方法的探索。1　肝再生增强因子的基因及蛋白质结构1994年Ｈａｇｉｙａ等[3 ] 首次克隆的大鼠ＡＬＲ的ｃＤＮＡ序列以及以后克隆的小鼠[4 ] 、大鼠[5,6] 和人[7～ 9] 的ＡＬＲ的ｃＤＮＡ基因序列 ,都认为不同种属的ＡＬＲ都是由 12 5个氨基酸残基组成的 ,其开放读码框架 (ＯＲ…     

人肝再生增强因子基因组DNA的克隆化与序列分析

目的 克隆人肝再生增强因子（ａｕｇｍｅｎｔｅｒ ｏｆ ｌｉｖｅｒ ｇｅｇｅｎｅｒａｔｉｏｎ,ＡＬＲ）的基因组ＤＮＡ,并进行序列分析。方法 采用人肝再生增强因子ｃＤＮＡ及其编码序列, 核苷酸序列数据库（ＧｅｎＢａｎｋ）以Ｂｌａｓｔ为工具进行核苷酸同源性比较分析,寻找相应的ＡＬＲ基因组ＤＮＡ序列。结果 从ＧｅｎＢａｎｋ核苷酸序列数据库中寻找到ＡＬＲ基因组ＤＮＡ全序列,由１８１３个核苷酸组成。     

O139,O1群霍乱弧菌分子遗传特征研究

目的探讨Ｏ１３９群、Ｏ１群霍乱弧菌分子遗传特征及其相互关系。方法用聚合酶链反应（ＰＣＲ）、ＤＮＡ序列分析及随机扩增多态性ＤＮＡ（ＲＡＰＤ）,检测了３株Ｏ１３９群、３株Ｏ１群ＥｌＴｏｒ生物型和２株古典生物型霍乱弧菌。结果３株Ｏ１３９群和５株Ｏ１群霍乱弧菌均扩增出５６６ｂｐ的ＤＮＡ片段,ＤＮＡ序列源于霍乱肠毒素（ｃｔｘ）Ａ２Ｂ基因。ＲＡＰＤ产生的两种ＤＮＡ指纹图谱一致显示Ｏ１３９群与Ｏ１群ＥｌＴｏｒ生物型遗传特征极其相似,与古典生物型略有区别。结论Ｏ１３９群与Ｏ１群ＥｌＴｏｒ型存在密切的种系进化关系,前者可能由Ｏ１群ＥｌＴｏｒ生物型进化而来。     

人肝再生增强因子在毕赤酵母中的表达

从人胎肝ｃＤＮＡ文库中克隆出人肝再生增强因子 (ｈｕｍａｎａｕｇｍｅｎｔｅｒｌｉｖｅｒｒｅｇｅｎｅｒａｔｉｏｎｈＡＬＲ) ,并实现ｈＡＬＲ在毕赤酵母中的表达。利用ＰＣＲ从人胎肝ｃＤＮＡ文库扩增出与文献报道序列一致的ｈＡＬＲｃＤＮＡ ,亚克隆到 ｐＰＩＣ9Ｋ的α因子下游 ,并置于ＡＯＸ1启动子控制下 ,构建成含有ｈＡＬＲ基因的酵母表达载体。转化酵母细胞后 ,挑选出能生长在Ｇ4 18浓度为 4 .0ｍｇ/ｍｌ,Ｈｉｓ 的重组酵母 ,利用ＰＣＲ鉴定是否插入ｈＡＬＲ基因。重组酵母表达在甲醇培养基中进行 ,并利用ＳＤＳ ＰＡＧＥ分析ｈＡ…     

cDNA sequence of rat liver fructose-1,6-bisphosphatase and evidence for down-regulation of its mRNA by insulin.

A coding-length clone of rat liver fructose-1,6-bisphosphatase (EC 3.1.3.11) was isolated by immunological screening of a cDNA library in lambda gt11. Its identity was verified by comparing the deduced amino acid sequence with that obtained by direct sequencing of a complete set of CNBr and proteolytic peptides from the purified protein. The enzyme subunit is composed of 362 amino acids and has N-acetylvaline as the amino-terminal residue. The cDNA, 1255 base pairs (bp) long, consisted of 1086 bp of coding region, 15 bp of 5' untranslated sequence, and 154 bp at the 3' untranslated end. The 3' untranslated sequence contained a polyadenylylation signal (AATAAA) followed after 30 bp by a stretch of 7 adenines at the end of the clone. The deduced amino acid sequence was identical to the primary sequence of the protein and confirmed the alignment of five nonoverlapping peptides. It also confirmed the 27-residue extension, unique to the rat liver subunit, ending with a carboxyl-terminal phenylalanine. RNA blot analyses using the radiolabeled liver cDNA as a probe revealed a single band of fructose-1,6-bisphosphatase mRNA, 1.4 kilobases long, in liver and kidney but not in nongluconeogenic tissues. Fructose-1,6-bisphosphatase mRNA was increased 10-fold in livers from diabetic rats and was reduced to control levels after 24 hr of insulin treatment, suggesting that the changes in enzyme activity observed in diabetes and after insulin treatment are due to alterations in mRNA abundance.     

Molecular cloning of human angiotensinogen cDNA and evidence for the presence of its mRNA in rat heart

Human angiotensinogen cDNA clone was isolated from a liver cDNA library using a 32-nucleotide-long, synthetic oligonucleotide. The cDNA insert was 1,030 bp long and coded for the secretory and biologically active angiotensin II regions of the angiotensinogen molecule. The RNA from rat liver, brain, and heart was analyzed by the Northern hybridization procedure using nick translated angiotensinogen cDNA as a probe. In addition to liver, the angiotensinogen mRNA is present in the brain and the heart. The angiotensinogen mRNA in the heart is at least fourfold to fivefold more abundant as compared with the liver. We also provide evidence that angiotensinogen mRNA is present in the rat atria and right ventricle but not detectable in the left ventricle. The size of the angiotensinogen mRNA is the same from all three of the tissues, as judged by their electrophoretic mobilities.     

Cloning and expression profile of FLT3 gene during progenitor cell-dependent liver regeneration

BACKGROUND AND AIM: The liver has a unique capacity to regenerate upon exposure to viral infections, toxic reactions and cancer formation. Liver regeneration is a complex phenomenon in which several factors participate during its onset. Cellular proliferation is an important component of this process and the factors that regulate this proliferation have a vital role. FLT3, a well-known hematopoietic stem cell and hepatic lineage surface marker, is involved in proliferative events of hematopoietic stem cells. However, its contribution to liver regeneration is not known. Therefore, the aim of this study was to clone and examine the role of FLT3 during liver regeneration in rats. METHODS: Partial cDNA of rat homolog of FLT3 gene was cloned from thymus and the tissue specific expression of this gene at mRNA and protein levels was examined by RT-PCR and Western blot. After treating with 2-AAF and performing hepatectomy in rats to induce progenitor-dependent liver regeneration, the mRNA and protein expression profile of FLT3 was investigated by real-time PCR and Western blot during liver regeneration. In addition, cellular localization of FLT3 protein was determined by immunohistochemistry. RESULTS: The results indicated that rat FLT3 cDNA has high homology with mouse and human FLT3 cDNA. It was also found that FLT3 is expressed in most of the rat tissues and during liver regeneration. In addition, its intracellular localization is altered during the late stages of liver regeneration. CONCLUSION: The FLT3 receptor is activated at the late stages of liver regeneration and participates in the proliferation response that is observed during progenitor-dependent liver regeneration.     

Polysome immunoprecipitation of phenylalanine hydroxylase mRNA from rat liver and cloning of its cDNA.

The mRNA for phenylalanine hydroxylase (phenylalanine 4-monooxygenase, EC 1.14.16.1) has been purified from total rat liver mRNAs, of which it constitutes less than 0.25%, to greater than 10% purity in a single step by specific polysome immunoprecipitation. The purified mRNA was used for synthesis and cloning of its cDNA. Recombinant colonies containing phenylalanine hydroxylase DNA sequences were identified by differential hybridization, hybrid-selected translation, and blot hybridization analysis. The rat cDNA clone was capable of hybridizing with human phenylalanine hydroxylase mRNA, which will permit the isolation of the corresponding human gene for analysis of phenylketonuria, a hereditary disorder in phenylalanine metabolism that causes permanent mental retardation in humans.     

High expression of human augmenter of liver regeneration in E.coli

ＩＮＴＲＯＤＵＣＴＩＯＮＨｅａｔｓｔａｂｌｅｈｅｐａｔｏｃｙｔｅｓｔｉｍｕｌａｔｏｒｙａｃｔｉｖｉｔｙｈａｓｂｅｅｎｄｅｓｃｒｉｂｅｄｉｎｔｈｅｌｉｖｅｒｏｆｗｅａｎｌｉｎｇｒａｔｓａｎｄｐｉｇｓ．Ｔｈｉｓｇｒｏｗｔｈｆａｃｔｏｒｉｓｃａｌｅｄｈｅ...     

Isolation and sequence determination of a cDNA clone for rat peroxisomal urate oxidase: liver-specific expression in the rat.

Urate oxidase (UOxase; urate:oxygen oxidoreductase, EC 1.7.3.3), which catalyzes the oxidation of uric acid to allantoin, is present in most mammals but is absent in humans and certain primates. A cDNA clone for UOxase containing an insert of 1.3 kilobases (kb) was isolated from a lambda gt11 cDNA library prepared from rat liver mRNA. This recombinant clone with a 1283-nucleotide insert has sequence for 97% of the coding region together with 401 nucleotides of the 3'-untranslated region of the mRNA. The identity of UOxase cDNA clone was verified by analyzing the fusion protein, immunocytochemical localization with epitope-selected antibody, and hybrid-select translation analysis and by comparing sequences of four CNBr-cleaved peptides of the protein. Blot analysis revealed that the probe hybridizes to a single 1.5-kb mRNA species in the rat liver and a transplantable hepatocellular carcinoma. No UOxase mRNA was detected in 11 nonhepatic tissues of rat, suggesting tissue specificity of expression of this UOxase gene. Blot analysis of RNA from livers of rats treated with a peroxisome proliferator showed 2- to 3-fold increase in UOxase mRNA content, whereas the fatty acyl-CoA oxidase mRNA increased over 30-fold. Southern blot analysis of restriction enzyme digests of rat DNA suggests that there is a single copy of UOxase gene. Analysis of human genomic DNA revealed restriction fragments that are homologous to rat UOxase cDNA, although no UOxase mRNA was detected in human liver.     

Expression of plasma glutathione peroxidase in human liver in addition to kidney, heart, lung, and breast in humans and rodents.

We analyzed the expression of plasma glutathione peroxidase (GSHPx-P) messenger RNA (mRNA) in mouse, rat, and human tissues, using a human GSHPx-P cDNA clone as the probe. Unlike the classical cellular glutathione peroxidase (GSHPx-1), GSHPx-P expression appears to be tissue-specific. In the mouse and rat, kidney expresses an mRNA at a high level detected with the human probe. A signal is also detected in mRNA isolated from mouse and rat heart, rat cardiac myocytes, mouse lung, epididymis, and the mammary gland of midpregnant mice. No signal is detected in mRNA isolated from mouse and rat liver, mouse brain, uterus, and testis. In human tissues, an mRNA hybridizing to GSHPx-P cDNA is present in liver, as well as kidney, heart, lung, breast, and placenta. We have shown that human kidney expresses a GSHPx-P mRNA, and not a GSHPx-P-like message, by isolating a cDNA clone from a human kidney library in lambda gt11. From the 412-nucleotide partial sequence of the kidney cDNA, which codes for the 40-170 amino acids of GSHPx-P including the TGA codon for selenocysteine, we found complete sequence identity of the kidney cDNA with GSHPx-P isolated from placenta. The expression of GSHPx-P mRNA in cell lines was also studied. There is some correlation of the expression of GSHPx-P in these cell lines with that in normal tissues. Cell lines that expressed GSHPx-P mRNA or protein included the human hepatocarcinoma HepG2, Hep3B cells, human kidney carcinoma A498 cells, and the human breast cancer SK-BR-3, T47D, MDA-MB-231, and AdrrMCF-7 cells. Cell lines that did not express GSHPx-P included human choriocarcinoma BeWo cells, human breast cancer MCF-7, ZR-75-1, and Hs578T cells, and mouse hepatoma Hepa-1 cells.