鉴定9个新的RHD基因mRNA可变剪接体

为了研究各种RHD基因mRNA可变剪接体的基因结构, 应用逆转录聚合酶链反应(RT-PCR)检测正常人脐血样本RHD mRNA, 对RHD cDNA进行TA克隆和序列分析, 对各可变剪接体的剪接位点进行DNA序列分析, 并将RHD mRNA进行表达序列标签(ESTs)分析。结果在28个阳性克隆中, 除全长RHD cDNA外, 共检测到12种(包括9种新的)RHD可变剪接体, 发现外显子遗漏、5′和3′剪接位点变异3种剪接形式, 涉及外显子2~9, 其中6种新的剪接体同时存在RHD和RHCE基因同源杂交现象。ESTs分析还检索到内含子保留形式的剪接体。研究表明, RHD基因mRNA存在复杂的可变剪接机制, 除已报道的剪接体外, 检测到9种新的RHD可变剪接体, 并发现了可变剪接和同源杂交并存现象。     

人恶性疟杂合多肽抗原基因化学合成及克隆

本文报道用固相亚磷酰胺法合成人恶性疟杂合多肽抗原基因。基因全长为216bp,分为10个寡聚核苷酸片段分别合成,然后经T4 DNA连接酶按设计顺序连接成完整的杂合抗原基因,重组到噬茵体M13 mp 18 RF DNA内,转染大肠杆菌JM109。用分子杂交和酶切分析筛选出重组克隆体。经序列分析,证明所合成的人恶性疟杂合多肽抗原基因与所设计完全一致。     

小鼠Sry基因和Sox基因的克隆分离与鉴定

为了深入探讨性分化分子机制,构建了小鼠基因库以ＳＲＹ探针筛选,分离到了两组阳性克隆,一组含ＥｃｏＲⅠ／３．５ｋｂ,此片段中载有小鼠Ｙ染色体上的Ｓｒｙ基因,另一组含ＥｃｏＲⅠ／３．０ｋｂ或ＳａｌⅠ／３．０ｋｂ,此片段有小鼠Ｙ染色体之外的Ｓｏｘ基因。     

人凝血因子Ⅶ cDNA基因的克隆与鉴定

目的:克隆并鉴定人凝血因子ⅦcDNA基因。方法:采用反转录聚合酶链反应(RT-PCR)的方法,从人胎肝总RNA中扩增人凝血因子ⅦcDNA基因,将其克隆入pGEM-T载体,对阳性克隆进行序列测定和分析。结果:经RT-PCR扩增和克隆,获得了人凝血因子ⅦcDNA基因,经序列分析表明,所克隆的基因序列正确。结论:本试验成功克隆了人凝血因子ⅦcDNA基因,为重组人凝血因子Ⅶ的研究奠定了基础。     

Rh血型的基因、抗血清及表现型的命名

Rh血型的名称取自恒河猴学名Rhesusmacaque的前两个字母,因为在最初探索新血型物质的过程中发现,当把恒河猴的红细胞反复地注入家兔及豚鼠体内时,家兔和豚鼠的血清可使恒河猴的红细胞凝集,说明恒河猴的红细胞上含有某种抗原,遂命名为Rh抗原。以后又发现经免疫的家兔或豚鼠的血清还能使大部分人的红细胞凝集,表明这些人的红细胞上也含有Rh抗原,并称之为Rh阳性血型,还有一些人的红细胞不被这种抗血清所凝集,便称为Rh阴性血型,由Rh抗原所决定的这一血型系统就称为Rh血型。后来,随着对各种输血反应、新生儿溶血症以及血清学试验的研究进展,…     

人canstatin基因的克隆

为深化研究血管生成抑制剂canstatin的生物学性能和用于肿瘤生物治疗的可能性,针对canstatin cDNA序列设计引物,运用逆转录聚合酶链反应（RT－PCR）从人胚肝组织中钓取canstatin cDNA,将其克隆入pMND18-T载体中,通过酶切鉴定出重组体并测序分析,结果表明,获得684bp人canstatin基因,成功构建人canstatin cDNA 克隆载体pMB18-T/canstatin,为进一步进行canstatin蛋白表达及活性研究奠定了基础。     

抗菌肽-X基因的克隆及在大肠杆菌中的表达

用PCR技术获得抗菌肽—X基因、TNFα基因,与温度诱导的表达载体pRC连接成为重组表达载体,导入大肠杆菌TG1,通过温度诱导表达重组蛋白。将重组质粒转入不同的表达菌中进行表达,经SDS—PAGE选出E.coil BL21(DE3)为最佳表达的宿主菌。培养后,离心得菌体,经超声破碎离心得包涵体,溶解后用CNBr切割并透析,最后经CM52纤维素柱分离纯化得到有活性高纯度的抗菌肽—X。     

克隆与克隆动物基因相同吗

美籍华人牛满江教授,著名生物学家,１９１２年生于中国河北省。１９３２年考入北京大学,１９３６年毕业留校任助教,１９３７年在昆明西南联大任教。１９４４年由北大派出去美国斯坦福大学深造,１９４６年获博士学位。自此,先后在斯坦福大学,洛克菲勒医学研究所,后改名为洛克菲勒大学和坦普尔大学从事教学和研究工作。并获得坦普尔大学终身教授,名字被载入美国科学家名人录。从１９５３年起,他潜心于“核糖核酸在发育中独特功能”的研究,是这一研究领域的先行者。他创立了“外基因学说”,开创了人工培育新物种的新思路。获得“利利学术”及“古根海姆”奖。１９７０年被选为台湾中央研究院院士。牛教授身居海外,对祖国科学教育事业极为关注,１９７８年,首先接收中科院派去美国的访问学者,打破中美学术交流的禁令,１９８０年中科院授予他科学顾问,中国各地的大学及科研机构授予他名誉教授、顾问头衔者３０多个。近来,为促进中国生命科学的研究和教育事业的发展,在他主持下在北京成立了“牛满江基金会”,将为培养优秀人才,促进国际学术交流方面作更多的贡献。     

胰岛素样生长因子结合蛋白-7基因的克隆和表达

IGFBP-7是胰岛素样生长因子结合蛋白(IGFBP)家族中的一员,具有一些抑癌基因的特征。利用重组PCR技术获得了IGFBP-7基因的全长编码区序列,并将其克隆到pcDNA3．1／His—Myc真核表达载体中,得到重组质粒pcDNA3．1／His—Myc—IGFBP-7。将该重组质粒瞬时转染人胚胎肾293T细胞,Westem-blot分析表明,IGFBP-7在293T细胞中获得了表达,为进一步研究IGFBP-7的功能奠定了基础。     

An hypothesis about the selection of the Rh negative individuals

We have taken into account the possibility of identifying the cause of the selection of Rh negative individuals during the Paleolithic period, referring to the known data on Rh positive and Rh negative phenotype and the pathogenic agents inside the red blood cells. After having narrowed down the field, identifying the Babesia divergens as the most probable responsible agent for the selection of the Rh negative individuals, we discuss the epidemiologic mechanism which may have intervened, and we evaluate the likelihood of this hypothesis in relation to the available data. We conclude that the following sequence is possible: Babesia divergens, Ixodes ricinus, Ursus spelaeus and/or arctus, caves, Rh negative selection. This would appear to be confirmed by the coinciding of the distribution areas of Rh negative individuals, Ursus spelaeus and arctus, Ixodes ricinus and Babesia divergens. We have also mentioned the possibility that the extinction of Neanderthal Man had the same cause as the selection of the Rh negative individuals.     

Cloning and characterization of the first amphibian bradykinin gene

More than ten bradykinin-related peptides and their cDNAs have been identified from amphibians, but their genes are unknown. In present study, four cDNAs encoding one, two, four and six copies of bradykinin-related peptides were cloned from the frog (Odorrana grahami) skin cDNA library, respectively. Three bradykinin-related peptides (bradykinin, Thr6-bradykinin, Leu5Thr6-bradykinin) were deduced from these four cDNA sequences. Based on the cDNA sequence, the gene sequence encoding an amphibian bradykinin-related peptide from O. grahami was determined. It is composed of 7481 base pairs including two exons and two introns. The first exon codes signal peptide and the second exon codes acidic spacer peptide and Thr6-bradykinin. The promoter region of the bradykinin gene contains several putative recognition sites for nuclear factors, such as SRY, GATA-1, LYF-1, DeltaE, CDXA, NKX-2.5, MIF1 and S8. The current work may facilitate to understand the regulation and possible functions of amphibian skin bradykinin-related peptides.     

人参多肽基因的化学合成和克隆

 本文报道了具有降血糖作用的人参多肽基因的设计及用固相亚磷酰胺法的合成。再以质粒pUC19作为克隆载体,以大肠杆菌JM101为受体菌,克隆了人参多肽基因,并成功地获得了克隆菌株。     

Cloning and characterization of a gene encoding wheat starch synthase I

 A cDNA clone, and a corresponding genomic DNA clone, containing full-length sequences encoding wheat starch synthase I, were isolated from a cDNA library of hexaploid wheat (Triticum aestivum) and a genomic DNA library of Triticum tauschii, respectively. The entire sequence of the starch synthase-I cDNA (wSSI-cDNA) is 2591 bp, and it encodes a polypeptide of 647 amino-acid residues that shows 81% and 61% identity to the amino-acid sequences of SSI-type starch synthases from rice and potato, respectively. In addition, the putative N-terminal amino-acid sequence of the encoded protein is identical to that determined for the N-terminal region of the 75-kDa starch synthase present in the starch granule of hexaploid wheat. Two prominent starch synthase activities were demonstrated to be present in the soluble fraction of wheat endosperm by activity staining of the non-denaturing PAGE gels. The most anodal band (wheat SSI) shows the highest staining intensity and results from the activity of a 75-kDa protein. The wheat SSI mRNA is expressed in the endosperm during the early to mid stages of wheat grain development but was not detected by Northern blotting in other tissues from the wheat plant. The gene encoding the wheat SSI (SsI-D1) consists of 15 exons and 14 introns, similar to the structure of the rice starch synthase-I gene. While the exons of wheat and rice are virtually identical in length, the wheat SsI-D1 gene has longer sequences in introns 1, 2, 4 and 10, and shorter sequences in introns 6, 11 and 14, than the corresponding rice gene. Received: 5 June 1998 / Accepted: 29 September 1998     

Cloning and characterization of the human beta-glucuronidase gene

We have isolated a cosmid clone that contains GUSB, the human gene encoding beta-glucuronidase. The 21-kb gene contains 12 exons ranging from 85 to 376 bp in length. Exon 6 corresponds to the 153-bp deletion in the shorter of two types of cDNAs reported earlier, supporting the hypothesis that this cDNA arose by alternate splicing leading to exon skipping. The insert contains 4.2 kb of sequence upstream from the first exon and 6 kb 3' of the last exon. The clone expresses human beta-glucuronidase in stably transformed rat XCtk- cells. Comparison of the human gene organization with that recently reported for the murine beta-glucuronidase gene revealed that the intron/exon boundaries are identical. In the splice junctions, the most highly conserved regions are those identified as consensus sequences, and these are at least as highly conserved as bases encoding the translated portion of the gene.     

犬瘟热病毒核衣壳蛋白基因片段的克隆和表达

目的　构建pMal N重组表达载体 ,转化E .coliDH5α ,诱导表达犬瘟热病毒 (CDV)重组核衣壳 (N)蛋白。方法　采用RT PCR技术 ,从CDVRNA中扩增编码N蛋白的基因片段 ,通过连接反应 ,构建重组克隆载体和重组表达载体 ,转化感受态。E .coliDH5α细胞。通过IPTG诱导表达CDV重组N蛋白。结果　扩增出约 1 7kbCDV全长的主要结构蛋白N蛋白基因 ,通过PCR获得 5 36bpN蛋白基因片段。将N蛋白基因片段克隆入原核表达载体pMal C2 ,表达产物麦芽糖结合蛋白 (MAP)与N蛋白的融合蛋白的相对分子质量约 6 0× 10 3,与预期大小一致。结论　构建的pMal N重组载体所表达的CDVN蛋白为进一步研究CDV的特异、敏感的抗体检测方法打下基础     

大麻哈鱼基因文库的构建及其生长激素基因的克隆

本文主要论述了黑龙江省特有鱼种,大麻哈鱼全基因文库的构建方法以及从所构建的1.9×10~(?)噬菌体成斑单位/ugDNA基因文库中克隆出生长激素基因片段的研究结果,作者使用DM-BL3入噬菌为载体,将大麻哈鱼肝总DNA的15-20Kb片段重组到载体中,再包装成新的重组噬菌体,以含虹鳟鱼生长激素DNA部分序列的PAF51为探针,经[~(12)P]dCTP标记后,与噬菌斑杂交,自显影,获得3个阳性斑,复筛获得90％以上阳性斑,酶切回收生长激素基因片段,实验证明,已成功地获得大麻哈鱼生长激素基因片段,可供进一步亚克隆或转移研究使用。     

米根霉菌ldhL基因的克隆及其在大肠杆菌中的表达

以米根霉菌基因组DNA为模板,根据GenBank上已公布的米根霉L-乳酸脱氢酶基因(ldhL)序列设计特异性引物,PCR扩增得到963 bp的DNA片段,经序列分析后将其亚克隆到原核表达载体pET30a上,构建成重组质粒pET30a-ldhL.将pET30a-ldhL转化到BL21感受态细菌中,经IPTG诱导表达后进行SDS-PAGE分析,可见约43 kD的与预期大小一致的目的蛋白条带,结果表明ldhL基因在大肠杆菌中进行了表达,经酶活分析产物的酶活力为98 U/mL,证明了表达产物具有预期的酶活性,这为进一步研究利用乳清为发酵原料高产L-乳酸的米根霉基因工程菌株奠定了基础.