抗人血管内皮生长因子(VEGF)单克隆抗体轻链可变区基因的克隆和序列分析

为构建和表达抗人血管内皮生长因子（ＶＥＧＦ）工程抗体奠定基础，从分泌抗人ＶＥＧＦ抗体的杂交瘤细胞株中提取总ＲＮＡ，利用反转录－聚合酶链反应方法，克隆抗人ＶＥＧＦ单克隆抗体轻链可变区基因，将其重组入ｐＥＧＭＲ－ＴＶｅｃｔｏｒ测序载体测序。结果表明轻链可变区序列全长３３３ｂｐ，编码１１１个氨基酸，通过国际联机检索及Ｋａｂａｔ库扫描证实，该轻链可变区符合小鼠免疫球蛋白轻链可变区基因特征，同源性高达８９．３％，归属小鼠轻链可变区基因第Ⅲ亚组，由ＶＬ－Ｊκ２重排产生     

抗人血管内皮生长因子（VEGF）单克隆抗体重链可变区基 …

克隆并分析人血管内皮生长因子单克隆抗体重链可变区基因。方法从分泌抗人ＶＥＧＦ单抗的杂交瘤细胞株中提取总ＲＮＡ，利用逆转录－ＰＣＲ方法，克隆抗人ＶＥＧＦ单克隆抗体重链可变区基因，将其重组ｐＥＧＭ＾Ｒ－ＴＶｅｃｔｏｒ测序载体测序。     

抗人血管内皮生长因子（VEGT）单克隆抗体轻链可变区基因的克 …

为构建和表达抗人血管内皮生长因子工程抗体奠定基础，从分泌抗人ＶＥＧＦ抗体的杂交瘤细胞株中提取总ＲＡＮ，利用反转录－聚合酶链反应方法，克隆抗人ＶＥＧＦ单克隆轻链可变区基因，将其重组入ｐＥＧＭ＾Ｒ－ＴＶｅｃｔｏｒ测序载体测序。     

抗变形链球菌SAⅠ/Ⅱ单抗重链可变区基因克隆及序列分析

从抗变形链球菌ＳＡⅠ／Ⅱ单抗杂交瘤细胞系２Ｂ１２Ｆ６中扩增克隆重链可变区基因,方法采用ＰＣＲ技术和基因工程技术,扩增杂交瘤细胞系２Ｂ１２Ｆ６的重链可变区基因并克隆入载体ＰＵＣ１８,Ｓａｎｇｅｒ双脱氧链末端终止法测定核苷酸序列。     

小鼠釉原蛋白成熟肽编码区cDNA的克隆和部分序列分析

目的：克隆小鼠釉原蛋白（ＡＭＧ）成熟肽编码区基因。方法：用异硫氰酸胍一步法从昆明新生小鼠磨牙牙胚组织中抽提总ＲＮＡ，用Ｏｌｉｇｏ（ｄｔ）作引物逆转录合成牙胚ｃＤＮＡ，然后利用ＰＣＲ方法，从ｃＤＮＡ中扩增出小鼠釉原蛋白成熟区的基因片段（约６７０ｂｐ），将所得基因片段插入ｐＢＳ质粒载体，转化到大肠杆菌ＤＨ５α后挑选阳性克隆，提取重组质粒ＤＮＡ，通过限制性酶切和部分核苷酸序列分析鉴定阳性克隆。结果：重组质粒ｐＢＳ－ＡＭＧ的酶切图谱和序列分析结果与国外文献报道一致。结论：克隆到小鼠釉原蛋白成熟肽编码区基因。     

巨型颈动脉体瘤切除后一期人造血管重建

巨型颈动脉体瘤切除后一期人造血管重建ＳＴＡＧＥＩＲＥＣＯＮＳＴＲＵＣＴＩＯＮＶＡＳＣＵＬＡＲＯＰＥＲＡＴＩＯＮＡＦＴＥＲＥＸＣＩＳＩＯＮＯＦＨＵＧＥＣＥＲＶＩＣＡＬＡＮＥＵＲＹＳＭ林朝生储琪东李跃俊余红梅作者单位：上海第二医科大学附属瑞金医院口腔科（...     

成骨蛋白—1成熟蛋白编码区cDNA克隆与序列分析

目的：克隆成骨蛋白－１（ＯＰ－１）成熟蛋白编码区基因。方法：用一步酸酚法从中国健康人胎盘组织中提取总ＲＮＡ，用Ｏｌｉｇｏ（ｄｔ）作引物逆转录合成胎盘ｃＤＮＡ文库，然后利用ＰＣＲ的方法，从ｃＤＮＡ文库中扩增出编码人ＯＰ－１成熟区的基因片段，将所得基因片段插入ｐＧＥＭ－３ｚｆ（＋）载体，转化大肠杆菌后挑选阳性克隆，提取双链ＤＮＡ模板，用ＡＢＩＤＮＡ自动测序仪进行序列测定分析。结果：克隆和测序结果与国外文献报道一致。结论：在国内第一次克隆到ＯＰ－１的成熟蛋白编码区基因。     

头颈部肿瘤累及颈动脉的外科治疗

头颈部肿瘤累及颈动脉的外科治疗ＳＵＲＧＥＲＹＯＦＨＥＡＤＡＮＤＮＥＣＫＴＵＭＯＲＩＮＶＯＬＶＩＮＧＴＯＣＡＲＯＴＩＤＡＲＴＥＲＹ郑家伟综述邱蔚六张志愿审校作者单位：上海第二医科大学附属第九人民医院口腔颌面外科（２０００１１）头颈部恶性肿瘤发展至晚期，...     

人牙髓细胞Smad2基因MH2结构域的cDNA 克隆和序列测定

目的：从人牙髓细胞内克隆转化生长因子－β特异的细胞内信号转导基因Ｓｍａｄ２的功能性结构域———ＭＨ２结构域。方法：原代培养人牙髓细胞,从培养的细胞中提取总ＲＮＡ,逆转录合成ｃＤＮＡ第１条链;设计内、外侧两对引物,进行巢式ＰＣＲ,扩增Ｓｍａｄ２基因ＭＨ２结构域的基因片段;将所获得的基因片段定向插入ｐＢｌｕｓｃｒｉｐｔⅡＳＫ（＋）载体;转化大肠杆菌ＪＭ１０９,挑选阳性克隆并鉴定;用ＰＥ３１７－Ａ型自动测序仪进行核苷酸序列测定分析。结果：测序结果与国外从人肾ｃＤＮＡ文库中克隆的基因序列完全一致。结论：首次从人牙髓细胞中克隆成功Ｓｍａｄ２基因的ＭＨ２结构域,证实了Ｓｍａｄ２基因在人牙髓细胞中的表达;提示人牙髓细胞内存在Ｓｍａｄ２信号转导途径,ＴＧＦ－β对人牙髓细胞分化的调节可能是通过Ｓｍａｄ２信号转导途径实现的。     

口腔颌面部血管外皮肉瘤(附6例报告)

口腔颌面部血管外皮肉瘤（附６例报告）ＨＥＭＡＮＧＩＯＰＥＲＩＣＹＴＩＣＳＡＲＣＯＭＡＯＮＯＲＡＬＡＮＤＭＡＸＩＬＬＯＦＡＣＩＡＬＲＥＧＩＯＮ：ＡＲＥＰＯＲＴＯＦ６ＣＡＳＥＳ杜晓军沈言备黄晓峰血管外皮肉瘤ＨｅｍａｎｇｉｏｐｅｒｉｃｙｔｅＳａｒｃｏ－ｍａ...     

Cloning and sequencing of variable region gene of heavy chain of monoclonal antibody against SA I/II of Streptococcus mutans]

OBJECTIVE: To clone and sequence a immunoglobulin variable region of heavy chain (VH) from a mouse hybridoma 2B12F6, which produce monoclonal antibody against SA I/II of Streptococcus mutans. METHODS: The immunoglobulin variable region gene of heavy chain of 2B12F6 was amplified and cloned into pUC18 by using PCR technique and gene engineering technique, and then the gene sequence was analyzed by Sanger's method. RESULTS: The VH gene segment was 360 base pairs in length and coded 118 amino acids, and the homology of framework of VH gene and mouse VH gene published was 70%, which accorded with the feature of mouse VH gene. CONCLUSION: The VH gene gained from 2B12F6 could provide the possibility of construction of gene engineering antibody against SA I/II of Streptococcus mutans.     

615小鼠H-2K~k基因cDNA的克隆及序列测定和分析

目的:克隆615小鼠主要组织相容性复合体(MHC)Ⅰ分子抗原识别基因H-2Kk并测序分析,为转基因治疗提供目的基因。方法:采用RT-PCR法从615小鼠肝组织总RNA中获得114 kb的H-2Kk基因cDNA,将其定向连接至 PGEM3Zf(+)载体,转化E.coliJM109,限制性内切酶筛选出阳性克隆PGEM3Zf(+)-H-2KkcDNA,末端终止法完成 H-2KkcDNA的全序列测定。结果:测得的H-2KkcDNA序列与文献报道序列同源性高达99%,编码MHC I分子抗原识别部位的氨基酸残基的核苷酸序列完全相同。结论:成功克隆了615小鼠MHCⅠ分子抗原识别基因,获得了表达MHC Ⅰ 功能的目的基因。     

Derived protein and cDNA sequences of hamster amelogenin

Hamster enamel protein extracts were analyzed by RP-HPLC and the isolated fractions by SDS-and Western blotting using polyclonal antibodies against recombinant mouse amelogenin and anti-peptide antibodies against the mouse exon 4-encoded sequence. Total RNA was extracted from enamel organ epithelia and, using a 3′ rapid amplification of cDNA ends (3′ RACE) technique, the coding regions for three different amelogenin isoforms were cloned along with the 3′ non-coding region. DNA sequencing revealed that the hamster amelogenin isoforms are 180, 73 and 59 amino acids in length, respectively. The 59-residue amelogenin corresponds to the leucine-rich amelogenin protein (LRAP), the 73-residue amelogenin corresponds to LRAP with the inclusion of the exon 4-encoded sequence, while the 180-residue amelogenin is the most abundant amelogenin isoform. Edman degradation was performed on purified hamster amelogenin, which provided the amino acid sequence in the region encoded by the 5′ PCR amplification primer used in cloning. Therefore, the entire derived amino acid sequence of hamster amelogenin was revealed. The hamster amelogenin amino acid sequence was aligned with all its known homologues. Hamster differs from rat and mouse amelogenin at only three amino acid positions. Southern blot analysis using a panel of restriction enzymes gave the same pattern for hamster DNA obtained from males and females, suggesting that in hamster, as in mouse, amelogenin is expressed from a single gene located on the X chromosome.     

牙龈卟啉菌16S rRNA基因片段的克隆和序列测定

目的:对牙龈卟啉菌的16S rRNA基因片段进行克隆和序列测定,为临床检验该菌提供依据.方法:通过聚合酶链式反应,扩增出牙龈卟啉菌16S rRNA基因内一段DNA片段,回收后用T载体进行克隆并测序.结果:聚合酶链式反应扩增出一段198bp的DNA片段,测出的序列与Genbank中牙龈卟啉菌的16S rRNA基因片段吻合.结论:聚合酶链式反应能够扩增出牙龈卟啉菌部分16S rRNA基因片段,可用于临床牙龈卟啉菌的检测.     

小鼠Dlx-5 cDNA的克隆及序列测定

目的 :扩增小鼠Dlx 5的特异性片段 ,构建重组质粒并进行序列测定 ,为进一步的蛋白表达、抗体制备、基因转染等研究奠定基础。方法 :提取E14d小鼠的下颌弓组织总RNA ,以反转录cDNA为模板进行PCR反应 ,克隆Dlx 5至PMD 18T载体上并测序。结果 :从胎鼠下颌弓组织中克隆到Dlx 5基因片段约 869bp ,重组质粒经酶切显示获得正确重组子 ,测序结果与已知序列吻合。结论 :成功地从小鼠下颌弓中克隆到了Dlx 5基因 ,提示胎鼠的下颌弓组织中有Dlx 5基因的表达。     

小鼠PeriostinC端编码区cDNA克隆及其序列分析

目的：从小鼠牙周膜组织中克隆PerostinC末端编码区基因。方法：用异硫氰酸胍一步法从昆明成年小鼠牙周膜组织中抽提总RNA,用Oligo(dt)作引物逆转录合成cDNA,然后利用PCR技术,从cDNA中扩增出小鼠PeriostinC末端编码区的基因自然（约940bp）,将所得基因片段插入p RSET-B载体,转化大肠杆菌DH5α后随机挑选数个克隆,提取质粒DNA,通过限制性酶切和核苷酸序列分析鉴定阳性克隆。结果：重组质粒pRSET-B-Periostin的酶切图谱和序列分析结果与国外文献报道一致。结论：克隆到小鼠该蛋白C端编码区基因。     

Analysis of single nucleotide polymorphisms in the 5'-flanking region of tumor necrosis factor-alpha gene in Japanese patients with early-onset periodontitis.

BACKGROUND: Early-onset periodontitis (EOP) is considered to have a genetic basis, which has not been clearly defined. The tumor necrosis factor-alpha (TNF-alpha) gene polymorphism as one of the genetic factors may influence the expression of several chronic inflammatory diseases. The aim of the present study was to evaluate whether the polymorphisms in the 5'-flanking region of the TNF-alpha gene are associated with Japanese EOP patients. METHODS: Forty-six Japanese, generalized EOP (G-EOP) patients and 104 Japanese healthy subjects were identified according to established clinical criteria. Twenty healthy subjects were analyzed by nucleotide sequence to screen polymorphisms of the 5'-flanking region of the TNF-alpha gene. Then, all subjects were analyzed by polymerase chain reaction and sequence-specific oligonucleotide probe (SSOP) methods. RESULTS: We determined 5 single nucleotide polymorphisms at positions -1031 (T/C), -863 (C/A), -857 (C/T), -308 (G/A), and -238 (G/A) in the 5'-flanking region of the TNF-alpha gene. There were no significant differences in the genotype and allele frequency when we compared G-EOP patients to healthy subjects. Because the frequency of polymorphic alleles at positions -308 and -238 was very low in this study population, we demonstrated the existence of 4 detected haplotypes and 6 detected genotypes concerning 3 single nucleotide polymorphisms (-1031, -863, and -857). The frequency of the H1/H3 (TCC/TCT)-detected genotype tended to decrease in G-EOP patients compared to healthy subjects, but was not statistically significant. CONCLUSION: These findings suggest there is no significant association between polymorphisms in the 5'-flanking region of the TNF-alpha gene and susceptibility to G-EOP in Japanese patients.     

远缘链球菌GTF催化活性蛋白及其多克隆抗体的制备和鉴定

目的:在大肠杆菌中表达重组远缘链球菌葡糖基转移酶(glucosyltransferase,GTF)的催化活性区(catalytic region,CAT)蛋白并制备其多克隆抗体。方法:扩增远缘链球菌OMZ176基因组的CAT区的基因片段,克隆入pQE31载体诱导表达,鉴定表达产物。重组蛋白纯化后免疫小鼠制备多克隆抗体,ELISA测定抗体的效价,West-ern blotting检测抗体的特异性和亲和性。结果:重组质粒成功在大肠杆菌中表达,经抗His tag单克隆抗体免疫印迹法检测,有阳性条带出现,证实重组蛋白有抗原特异性。ELISA测定免疫小鼠所得抗CAT多克隆抗体效价可达到1∶5000。Western blotting检测证明该抗体有较好的针对CAT蛋白的专一性。结论:GTF区CAT基因原核表达质粒构建成功,表达的融合蛋白具有良好的抗原性。抗CAT多克隆抗体特异性和效价良好,能够满足针对CAT免疫印迹和细胞免疫组化检测等实验要求,为深入研究同时包含变形链球菌和远缘链球菌两种致龋菌的主要抗原的新型防龋DNA疫苗奠定了必要的物质基础。     

牙龈卟啉菌及其胶原酶基因遗传多样性的研究

目的 分析牙龈卟啉单胞菌细菌基因型及其重要毒力因子胶原酶基因PrtC的遗传异质性，了解细菌遗传变异与其致病性的相关性。方法 对从不同牙周炎患者口腔中分离出的牙龈卟啉菌进行DNA指纹分析，参考菌株为PgATCC33277。通过PCR反应，检测临床菌株中是否存在特异性的胶原酶基因片段（548bp）。并通过酶切鉴定和DNA序列分析，了解PrtC基因遗传多样性的变化。结果 80株牙龈卟啉菌共获得7种基因型（Ⅰ-Ⅶ）。所有24株临床菌株和参考菌株PgATCC3327中均扩增出特异性的胶原酶基因片段。对照菌株中没有得到相应的产物。4株细菌的序列分析结果与国外的报道相比较，发现一些基因序列存在差异，出现6个核苷酸碱基缺失。结论 临床分离的牙龄卟啉菌中可检测到多种基因型，且具有个体差异，这些菌株具有合成胶原酶的能力，不同菌株胶原酶基因之间存在遗传异质性的变化。