【作者】 杨天燕；

【导师】 王乃平；

【作者基本信息】 广西医科大学 ， 药理学， 2009， 博士

【摘要】 组胺(2-[4-imodazole]-ethylamine)是组胺酸在组胺酸脱羧酶(histidine decarboxylase, HDC)作用下形成的体内作用最广泛的单胺类物质之一,是I型超敏反应中的主要效应分子,组胺通过与其相结合的Hrh1(histamine receptor H1)参与机体内多种生理功能的调节。Hrh1是研究神经-内分泌-免疫-效应器轴的重要中介。Hrh1作为单拷贝基因,在大鼠基因组中定位于4号染色体,4q42区。直至最近的研究才发现,人类的Hrh1基因属于无内含子的结构基因。截至2009年4月,NCBI中报道的人类基因组无内含子基因约为180个,数量远远少于有内含子基因,但是,真核生物的无内含子基因在比较物种进化与基因组的遗传变异中起着重要的作用,近年来已逐渐引起人们的兴趣。大鼠Hrh1基因也是无内含基因,全长均为外显子。目前,针对Hrh1基因多态性与碱基突变的研究尚不多,国内未见有关大鼠Hrh1基因突变的报道。大鼠Hrh1基因与人基因组Hrh1基因具有较高的同源性,其蛋白结构相近,具有两段7个跨膜α螺旋结构域,氨基酸序列比对同源性约80%,因此我们选择大鼠Hrh1基因研究其基因组与转录产物mRNA的碱基突变,核苷酸多态性,为今后研究人类基因组Hrh1基因多态性,相关突变及与Hrh1表达产物(组胺H1受体)的遗传药理学与药物基因组奠定前期实验基础。针对我们的研究目的,本课题实验主要分为四个部分。第一部分实验,我们研究了大鼠Hrh1基因在大鼠全血基因组、大鼠脑组织转录产物mRNA以及大鼠肝组织转录产物mRNA中序列的碱基差异。主要实验操作方法为:从雄性SD大鼠中用碘化钾法提取全血基因组DNA,Trizole法提取大鼠脑组织与肝组织的总RNA。紫外分光光度法与琼脂糖凝胶电泳鉴定提取的核酸纯度与浓度合格后,以脑组织和肝组织的总RNA为模板,RevertAid? First Strand cDNA Synthesis Kits逆转录合成cDNA第一链。根据NCBI公布的大鼠Hrh1基因组DNA(gi: 62750804)及mRNA序列(gi: 220770),应用Vector NTI Suite 10.0软件,针对Hrh1的蛋白编码区序列设计克隆编码区全长的引物,为便于今后研究Hrh1基因的表达,在上下游引物的5’引入不同的限制性酶酶切位点。使用保真度极高的PrimeSTAR? HS DNA Polymerase,以所提取的大鼠全血基因组DNA、脑组织cDNA以及肝组织cDNA为模板,使用聚合酶链反应(PCR)方法克隆大鼠Hrh1编码区全长。PCR产物纯化回收试剂盒对PCR反应的产物进行纯化。用限制性内切酶双酶切法对基因组DNA、脑cDNA、肝cDNA PCR纯化产物与克隆载体pUC19进行双酶切。小量胶回收试剂盒进行酶切产物的纯化回收。T4 DNA Ligase分别将基因DNA,脑cDNA,肝cDNA酶切产物与pUC19载体进行粘性末端的连接,构建pUC19-Hrh1重组载体。用Inoue法制备大肠杆菌DH5α的超级感受态。将构建好的pUC19-Hrh1通过热激转化至DH5α的感受态细胞中,用IPTG,X-gal对转化的感受态进行蓝白筛选,用菌落PCR法快速筛选含有pUC19-Hrh1的阳性菌落。对筛选好的阳性菌落进行过夜培养,用小量质粒快速抽提纯化试剂盒进行质粒提取。提取的质粒首先进行凝胶电泳,根据质粒条带初步确定质粒的大小。以质粒为模板,进行PCR扩增,根据是否有特异性条带判定重组载体中是否含有目的基因。为保证筛选出的重组载体不含有目的片段的双克隆,以限制性内切酶对提取的质粒进行双酶切鉴定。从筛选合格的重组子中,我们选择了3个血基因的重组克隆子、3个脑cDNA的重组克隆子以及5个肝cDNA的重组克隆子送北京诺赛基因组研究中心有限公司进行测序。根据测序结果进行比对,我们发现血基因组3个克隆子与脑cDNA的3个克隆子、肝cDNA的1个克隆子与NCBI中公布的大鼠Hrh1基因组DNA(gi: 62750804)序列100%一致,但与mRNA(gi: 220770)序列的一致性为99.7%,存在4个碱基差异。而肝cDNA组的另4个克隆存在5个位点的碱基突变,分别是237位、928位、1041位、1210位以及1342位。通过分析氨基酸的三联体密码,我们发现928位与1342位突变可导致编码的氨基酸发生改变。928位为蛋氨酸(Met)→缬氨酸(Val),即Val代替了Met;1342位为Val→Met。根据NCBI公布的大鼠Hrh1的cSNP位点,仅237位(rs8155549)的C/T多态与我们的测序结果相符,其余的4个突变位点,未见有相关的报道。因此,我们推测:①大鼠Hrh1基因中,存在有新的cSNP位点。②Hrh1在大鼠体内不同组织中转录时(如脑组织与肝组织),有可能存在RNA水平上的突变,即RNA编辑。在第二部分实验中我们主要研究不同抗凝剂对全血基因组DNA提取的纯度和得率,尤其是下游的基因扩增的影响。在第一部分实验中,我们发现当使用肝素钠抗凝时,紫外分光光度法与琼脂糖凝胶电泳检测基因组DNA得率与纯度都令人满意,但PCR扩增时,结果并不稳定,效果亦不理想。在排除多种因素并查阅了相关资料后,推测有可能是抗凝血中抗凝剂的使用对基因组DNA的扩增效率产生影响。考虑到EDTA对PCR反应的催化剂Mg2+的螯合作用,我们没有选择EDTA作为抗凝剂,而是选用枸橼酸钠与肝素作为抗凝剂,分别对大鼠动脉血与静脉血抗凝后,进行实验比较。设计了3对引物,引物1扩增β-actin(跨内含子设计,1104 bp);引物2扩增Hrh1基因的部分片段,468bp;引物3扩增Hrh1全长,1477bp。由实验结果可知,当使用肝素钠作为抗凝剂从全血中提取核酸进行基因扩增实验,扩增效率会受到明显的抑制,尤其扩增的目的片段较长(>1000 bp)时。扩增小片段时,肝素钠对PCR的扩增效率影响较小。而动脉血与静脉血的DNA扩增结果没有差异。根据这部分的实验结果,我们在提取血基因DNA时,选择使用枸橼酸钠作为抗凝剂,保证下游实验结果的稳定性。为了确定大鼠Hrh1基因的cSNP位点,证实在第一部分实验中的推测,我们进行了第三部分实验。这部分实验选择健康雄性SPF SD级大鼠7只,健康雌性SD大鼠6只,颈静脉取血,碘化钾法提取全血基因组DNA。紫外分光光法与琼脂糖电泳鉴定DNA的浓度与纯度。PrimeSTAR? HS DNAPolymerase高保真扩增大鼠Hrh1编码区全长,琼脂糖电泳鉴定PCR产物,小量胶回收试剂盒进行剩余PCR产物的纯化回收。纯化产物送北京诺赛基因组研究中心有限公司进行测序。通过分析13只大鼠的基因组Hrh1序列测序峰图,我们发现在大鼠Hrh1 CDS全长1461 bp中,共有4个cSNP位点,分别为:237位C/T多态;928位A/G多态;1041位C/T多态;1342位G/A多态。并对13例样本4个cSNP位点用SPSS 15.0进行了频率统计。截至2009年4月,NCBI中对大鼠Hrh1编码区报道的cSNP位点共为3个,分别为rs8153540(102位A/C多态)、rs8155549(237位C/T多态)以及rs8153541(1404位C/G多态)。本课题研究发现的4个cSNP位点仅237位C/T多态性cSNP与NCBI所报道rs8155549相符,其余3个cSNP位点(928位、1041位、1342位)NCBI均未见报道,而NCBI报道的rs8153540、rs8153541本研究中未发现多态性。我们发现的大鼠Hrh1基因编码区内4个cSNP位点中,237位与1041位C/T多态引起三联体密码子的改变不会影响编码氨基酸的改变。但928位A/G多态与1342位G/A多态能造成三联密码子编码的氨基酸发生改变,是错义突变。我们根据NCBI中公布的人类基因组Hrh1基因DNA序列、mRNA序列;大鼠Hrh1基因DNA序列、mRNA序列;小鼠Hrh1基因DNA序列、mRNA序列,应用Vector NTI Suite 10.0与Lasergene 7.0进行分析比对,发现大鼠Hrh1 cSNP位点与人类基因组Hrh1 cSNP位点,小鼠Hrh1 cSNP有着相似的发生区域。使用NCBI的Protein Blast功能进行了Hrh1基因所编码的蛋白结构预测,可知人类、大鼠、小鼠的Hrh1蛋白结构极为相似,都存在有2段7个跨膜α螺旋结构域,区间均为45～225AA和390～470AA。用Lasergene 7.0进行Hrh1编码蛋白多肽链的二级结构预测表明928位和1342位cSNP可导致编码蛋白多肽链310与448位氨基酸的改变。从预测分析可发现,氨基酸改变后会导致α区域与亲水性的改变。结果提示我们,突变引起编码多肽链一级结构的改变,最终导致翻译的蛋白质活性的改变,从而影响受体蛋白的特性和功能。为今后进行Hrh1 cSNP相关的遗传药理学与药物基因组学等实验确定相应的检测cSNP的方法,在第四部分实验中,我们探索了等位基因特异性扩增法(alleles specific amplification,ASA)检测928与1342位cSNP。选择第三部分实验中通过测序确定序列的1号(928 A/G, 1342 G/A)、2号(928 G/G, 1342 A/A)、8号(928 A/A, 1342 G/G)大鼠做为测定样本。ASA PCR引物的设计至关重要。针对928位(A/G)和1342位(G/A)cSNP位点,应用Vector NTI Suite 10.0软件设计引物。2个位点的引物都是在下游引物3’端设计相应的碱基探针,与探针相邻的碱基设计成错配。提取全血基因组DNA,高保真扩增Hrh1全长,PCR产物纯化回收,稀释后作为ASA PCR的反应模板。选择第一部分实验中通过测序确定的重组质粒Blood 1(928 A, 1342 G)和Liver 1(928 G, 1342 A)做为阳性对照。实验结果表明,PCR结果与预期结果一致,证明所设计ASA探针引物完全可行,建立ASA PCR反应体系可靠,为今后对大鼠的Hrh1基因进行分型,进行相应的Hrh1不同基因表型、遗传药理学与药物基因组学等相关实验奠定了前期的基础。更多还原

【Abstract】 Histamine is derived from the decarboxylation of the amino acid histidine, a reaction catalyzed by the enzyme L-histidine decarboxylase. It is one of monamines which have the most extensively influences on the functions of the body. Histamine is the major response factor in hypersensitivity type I. Mediated by Hrh1 ( histamine receptor H1 ) , histamine regulates comprehensive physiological function in eucaryotic organism. Therefore, Hrh1 is an important agency to study the nerve-incretion-immunity-effector axis.As a monocopy gene, Hrh1 is localized at Rattus norvegicus chromosome 4, 4q42 region. Homo Sapiens Hrh1 gene was thought to be intronless until recently. Up to April 2009, the numbers of human’s intronless gene reported at NCBI site were about 180, far less than the number of human’s intron gene. But eukaryotic intronless genes are important datasets for comparative genomics and evolutionary studies, and have evoked interesting of researchers. Rattus Hrh1 is also intronless gene and its full length is entire exon.There are few studies about the Hrh1 gene polymorphism and base mutation so far, and there is no report about the mutation of Rattus Hrh1 gene in domestic research. Rattus Hrh1 gene is highly consensus with human genomic Hrh1 gene. Their protein structures are very similar: both have two 7-transmembraneαhelix regions, in which homologous amino acid sequence is about 80%. According to this fact, we chose Rattus Hrh1 gene to study its base mutation and nucleotide polymorphism in genomic DNA and DNA transcript (mRNA), to lay the experimental foundation of polymorphism, relevant mutation, pharmacogenetics, and pharmacogenomics about human Hrh1 gene and its express product (histamine H1 receptor) in the future.The subject study is mainly in four parts for the purpose of our investigation.In experiment part I, we studied the sequence variation of rattus Hrh1 gene from different origins respectively, i.e. genomic DNA extracted from rat whole blood, transcript mRNA in rat brain tissue and transcript mRNA in rat hepatic tissue.We extracted a male SD rat genomic DNA from whole blood using KI method and extracted total RNA from rat brain tissue and hepatic tissue with Trizole. Ultraviolet spectrophotometry and agarose gel electrophoresis were used to detect purity and concentration of obtained nucleic acid. RevertAid? First Strand cDNA Synthesis Kits was used to synthesize first strand cDNA chain with brain total RNA and hepatic total RNA as reaction template. We apply Vector NTI Suite 10.0 to design the primers in compliance with coding sequence of rattus genomic Hrh1 DNA sequences (gi: 62750804) and mRNA sequence (gi: 220770) reported at NCBI site. According to the restriction enzymes digest site at 5’terminus in sense and antisense primer, we can conveniently construct the recombinant express vector in the future. PrimeSTAR? HS DNA Polymerase has very high fidelity, so we used it to clone the rat Hrh1 gene entire coding sequence by polymerase chain reaction with rat genomic DNA, brain cDNA, and hepatic cDNA as template. PCR purification kit was used to purify and recover the product of PCR. Double enzyme restriction was used to digest PCR product of genomic DNA, brain cDNA, and hepatic cDNA, and cloning vector pUC19 Gel extraction kit was used to purify and recover the product of double digestion. T4 DNA Ligase jointed the digestion product of genomic DNA, brain cDNA, hepatic cDNA and vector pUC19 respectively with their cohesive termini. By this, we construct pUC19-Hrh1 recombinant vector.The Inoue Method was used to prepare super competent of Ecoli DH5α. pUC19-Hrh1 recombinant vector was transformed into DH5αsuper competent by heat shock, and IPTG, X-gal was used to do a blue-white screen. Colony PCR was used to quickly identify the positive colony with pUC19-Hrh1. The selective positive colony was cultured overnight. Plasmid was extracted from the overnight culture with plasmid DNA extraction kit.Agarose gel electrophoresis was used to identify the size of plasmid according their band compared with the marker approximately. PCR amplification with plasmid as template was used to determine the target gene in selective recombinant. Double digestion was used to identify the plasmid to prevent selective plasmid from double cloning with target gene. We selected 3 recombinants of genomic DNA, 3 recombinants of brain cDNA, and 5 recombinants of hepatic cDNA, and committed these recombinants to SinoGenoMax Co., Ltd. for sequencing.By the consequence of sequencing, we found it was 100% consensus of 3 recombinants of genomic DNA, 3 recombinants of brain cDNA and 1 recombinant of hepatic cDNA with rattus Hrh1 genomic DNA (gi: 62750804), But only 99.7% consensus with mRNA (gi: 220770), with 4 bases difference. To our surprise, there were 5 base mutations within 4 recombinants of hepatic cDNA, at 237, 928, 1041, 1210, and 1342 locus. After the triplet code of amino acid was analyzed, we found the mutations at 928 and 1342 locus can lead to the change of translation amino acid with that valine replaces methionine at 928 locus and methionine replaces valine at 1342 locus. According to the report of cSNP of Rattus Hrh1 gene at NCBI site, only the C/T polymorphism (rs8155549) at locus number 237 coincides with our result. There was no relevant report about the rest 4 loci mutations. It is suggested that (1) there are new cSNPs in rattus Hrh1 gene; and (2) there may be mutate at RNA level, when Hrh1 gene transcripts in different organs (e.g. brain or liver) of the rat, that is also called RNA editing.In experiment part II, we mainly researched the influences of different anticoagulant to the purity and yield of DNA extracted from whole blood genome, especially subsequent PCR amplification. Because, in experiment part I, we extracted genomic DNA from blood anticoagulated with heparin sodium, yield and purify of genomic DNA, detected by ulraviolet spectrophotometry and agarose gel electrophoresis, had no problem. But the result of PCR amplification was not stable and satisfied. Referring to involved information and after excluding many factors, we speculated that anticoagulant in blood may impact PCR amplification efficiency. Considering EDTA as chelant can inhibit the Mg2+ to catalyze PCR, we didn’t choose it in this experiment, but use sodium citrate and heparin sodium as anticoagulant, to anticoagulate arterial blood and venous blood of rat respectively, and then perform the comparison.We designed 3 pairs of primers: Primers I amplifiesβ-actin (span across intron, 1104 bp); Primers II amplifies part of Hrh1 gene, 468bp; And primers III amplifies full length of Hrh1 gene, 1477 bp. From the result of experiments, we can make the conclusion that when genomic DNA from blood containing anticoagulant heparin sodium is extracted, the anticoagulant will inhibit PCR amplification efficiency, especially to amplify large size fragment. Heparin sodium inhibited amplification efficiency slightly while small size fragment was amplified. According to the experimental results, we extracted genomic DNA from blood with sodium citrate as anticoagulant, in order to guarantee the stability of sequent experiment.Part III of the experiments was cSNP detection in rattus Hrh1, to confirm our conclusion in experiment part I. We selected 13 healthy SPF SD rats, 7 males and 6 females, and phlebotomized the rats from jugular vein. Genomic DNA was extracted from whole blood by KI method. The purity and concentration of DNA samples were detected by the methods of ultraviolet spectrophotometry and agarose gel electrophoresis. PrimeSTAR? HS DNA Polymerase can be used to amplify full length of rattus Hrh1 gene coding sequence accurately. The product of PCR after agarose gel electrophoresis detection was purified and recovered with gel extraction kit. Purified product was committed to SinoGenoMax Co., Ltd. for sequencing.After studying the sequencing peak map, we discovered that there are 4 cSNPs in Rattus Hrh1 entire coding sequence which contains 1461 bp: 237 C/T polymorphism, 928 A/G polymorphism, 1041 C/T polymorphism, and 1342 G/A polymorphism. The statistical frequency of 4 cSNPs in 13 samples was analyzed by SPSS 15.0. Up to April 2009, the numbers of cSNP reported in rattus Hrh1 coding sequence at NCBI site are 3: (1) rs8153540 (102 A/C polymorphism);(2) rs8155549 (237 C/T polymorphism);and (3) rs8153541 (1404 C/G polymer- phism)。In the 4 cSNPs which we discovered, only cSNP 237 coincided with rs8155549 reported by NCBI, and the rest of other 3 cSNPs were not reported at NCBI site at the same time. We did not found the polymorphism about rs8153540 and rs8153541 which NCBI reported.In the 4 cSNPs, albeit alter triplet code changed by 237 C/T and 1041 C/T polymorphism will not change the coding amino acid. However, 928 A/G polymorphism and 1342 G/A polymorphism are missense mutation, and the polymorphism can alter the amino acid coded by changing triplet code。We applied Vector NTI Suite 10.0 and Lasergene 7.0 to analyze the homo sapience Hrh1 gene DNA sequence and mRNA sequence, Rattus norvegicus Hrh1 DNA sequence and mRNA sequence, and Mus Musculus Hrh1 DNA sequence and mRNA sequence, and discovered that the occurring region of cSNPs in Human Hrh1 gene, rattus Hrh1 gene and mus Hrh1 gene are resemble each other, although the numbers of human cSNPs in Hrh1 gene are much more than the others. By predicting the structure of Hrh1 gene coding protein, we found that their structures are very similar: all of them have two 7-transmem- brance-α-helix regions in the peptide chain, between 45-255AA and 390- 470AA.We predicted the secondary structure of Hrh1 protein peptide chain by Lasergene 7.0. The 928 and 1342 cSNPs can change the amino acid 310AA and 448AA in translated peptide chain. According to the secondary structure, alteration of amino acid can change the structure ofαregion and hydrophilicity. The result indicated that the activity of translated protein may be changed, resulting from amino acid mutation induced by the alteration of first structure of coding peptide chain, and finally impact the function and characteristic of receptor protein.For establish the approach to detect the cSNPs in rattus Hrh1 gene, which can carry out the experiments involved with pharmacogenetics and pharmacogenomics, in experiment part IV, we explored ASA (alleles specific amplification) PCR to detect the 928 and 1342 cSNPs. Sample rat 1 (928 A/G and 1342 G/A), rat 2 (928 G/G and 1342 A/A), and rat 8 (928 A/A and 1342 G/G), which already confirmed sequence of Hrh1 gene in experimental part III, were chose as detectable sample.Primers designed for ASA PCR are keys to the detection. Primers were designed by Vector NTI Suite 10.0 in accordance with 928(A/G) and 1342(G/A) polymorphisms. Primers of two positions were designed homologous base probe at 3’terminus, and the base next to 3’terminus probe was designed as mismatch. The genomic DNA was extracted from whole blood, the entire coding sequence of rattus Hrh1 gene was amplified, and the PCR product was purified and recoveried. The PCR product was diluted as template for ASA PCR later. Recombinant plasmid Blood 1 (928 A and 1342 G), and Liver 1 (928 G and 1342 A), for which, the sequences were confirmed in experimental part I, were chose as positive control.Experimental consequence showed that result of PCR coincide with our expectation, and proved that the probe primers for ASA PCR are qualify, and the system of ASA PCR reaction is reliable. Our results laid the experimental foundation for genotyping rat involved with Hrh1 gene and researching the mutant phenotype of Hrh1 gene, by the methods of pharmacogenetics and pharmacogenomics about human Hrh1 gene and its express product-histamine H1 receptor in the future.更多还原