日本血吸虫硫氧还蛋白谷胱甘肽还原酶基因克隆和序列分析

目的克隆日本血吸虫硫氧还蛋白谷胱甘肽还原酶（SjTGR）基因,并进行序列分析。方法制备日本血吸虫成虫mRNA,反转录合成cDNA,采用聚合酶链反应（PCR）技术,从日本血吸虫成虫cDNA中扩增出SjTGR基因片段。通过TA克隆技术将该基因片段克隆到pGEM-T载体中进行DNA序列测定和生物信息学分析。结果采用PCR技术成功地从日本血吸虫成虫cDNA中扩增出TGR基因片段,TA克隆后DNA序列分析显示该基因长度为1791bp,编码596个氨基酸残基,理论分子质量65kDa,生物信息学分析显示与曼氏血吸虫TGR氨基酸序列同源性为82%,含有吡啶核苷酸二硫化物氧化活性中心CVNVGC序列,在氨基端含有谷氧还蛋白特征性的活性位点CPFC基序。结论SjTGR基因克隆获得成功,为发展抗日本血吸虫新药及疫苗候选分子奠定了基础。     

编码日本血吸虫线粒体大亚基核糖体基因的亚克隆、测序及同源性分析

目的筛选日本血吸虫成虫 cDNA 文库,得到基因克隆并测序。方法体外将以阳性克隆为模板的 PCR 产物和 pGEM-T 载体连接,转染大肠杆菌 XL1-blue,经抗生素及生色底物 X-gal 初筛,再用限制性内切酶酶切法进一步鉴定为重组质粒后,DNA 自动测序仪测序。序列送blast 基因服务站进行同源性分析。结果构建三个含日本血吸虫 cDNA 基因片段的重组子,其中一个阳性克隆序列为编码日本血吸虫线粒体大亚基核糖体的基因序列。结论获得编码日本血吸虫线粒体大亚基核糖基因片段,为分析其作为候选重组疫苗分子的潜能打下基础。     

日本血吸虫10.6 kDa膜蛋白基因的克隆及表达

目的　寻找新的预防日本血吸虫感染疫苗候选分子。　方法　用具保护性的抗血吸虫膜单抗及免疫血清筛选日本血吸虫 c DNA文库 ,PCR扩增 c DNA插入片段 ,A - T连接法将筛选获得的 3个基因片段克隆到p GEM- T载体上 ,自动测序仪测序 ,序列送 BL AST基因服务站进行同源性分析。重新设计引物扩增编号为 B8克隆的开放阅读框碱基序列 ,先将其克隆入 p GEM- T载体质粒 ,再定向亚克隆入 p BK- CMV表达质粒 ,IPTG诱导表达后用 SDS- PAGE和 Western blotting分析表达产物。　结果　经双酶切及 PCR法均证实基因重组成功。其特异性表达产物约为 10 .6 k Da蛋白抗原。表达产物可被免疫血清及单抗识别。　结论　构建了编码日本血吸虫 10 .6k Da蛋白基因表达克隆。     

日本血吸虫SjPGK基因克隆和序列分析

目的　克隆日本血吸虫磷酸甘油酸激酶 (Sj PGK)编码基因片段 ,分析其核苷酸序列。方法　根据曼氏血吸虫磷酸甘油酸激酶 (Sm PGK) c DNA序列设计并合成一对引物 ,以日本血吸虫成虫总 RNA为模板 ,采用逆转录 -聚合酶链反应 (RT- PCR)特异性扩增 Sj PGK基因片段 ,将其克隆入p MD18- T载体中 ,经双酶切分析和 PCR鉴定 ,将阳性克隆进行脱氧核糖核酸序列测定 ;运用BL AST程序 ,将测序结果及其推导的编码氨基酸序列与 NCBI数据库在核苷酸水平和氨基酸水平进行同源性比较。结果　 RT- PCR特异性扩增出一条长约 830 bp的条带 ;重组质粒的双酶切和以其脱氧核糖核酸为模板的 PCR均可见一条与 RT- PCR产物相同的条带 ;脱氧核糖核酸序列测定和分析结果表明 :Sj PGK基因片段长为 830 bp,与 Sm PGK的核苷酸同源性为 85 % ,分值为 6 72 ;氨基酸同源性为 94 % ,分值为 4 73。结论　成功地克隆了 Sj PGK编码基因片段 ,为寻找日本血吸虫新的抗感染疫苗候选分子奠定基础     

日本血吸虫磷酸甘油酸激酶基因T/A克隆及序列分析

目的获取日本血吸虫磷酸甘油酸激酶(SjPGK)编码基因片段,分析其核苷酸序列,为日本血吸虫病疫苗研制提供候选抗原分子。方法根据曼氏血吸虫磷酸甘油酸激酶(SmPGK)cDNA序列设计1对引物,采用逆转录聚合酶链反应(RTPCR)方法,从日本血吸虫成虫总RNA中扩增SjPGK基因片段。利用T/A克隆法,将其克隆入pMD18T载体,经双酶切分析和PCR鉴定,将阳性克隆进行脱氧核糖核酸序列测定;运用Blast程序,将测序结果及其推导的编码氨基酸序列与NCBI数据库在核苷酸水平和氨基酸水平进行同源性比较。结果RTPCR特异性扩增出1条长约830bp大小的条带;经酶切和PCR鉴定表明所构建的质粒pMD18TSjPGK中含有所扩增的基因序列;脱氧核糖核酸序列测定和分析,SjPGK基因片段长为830bp,与SmPGK的核苷酸同源性为85%,分值为672;氨基酸同源性为94%,分值为473。结论成功克隆SjPGK编码基因片段,为进一步实验提供了条件。     

日本血吸虫脂筏蛋白的重组表达及其生物信息学分析

目的在大肠杆菌中原核表达、纯化日本血吸虫脂筏蛋白(rSjFLOTILLIN2),并对其进行生物信息学分析。方法以日本血吸虫成虫cDNA为模板扩增FLOTILLIN2基因片段,克隆至pET28a(+)质粒,经Xho I和EcoR I双酶切以及测序鉴定;重组质粒再转化入E. coli BL21,含重组质粒的菌株经IPTG诱导表达,采用SDS-聚丙烯酰胺凝胶电泳鉴定目的蛋白分子量大小。同时采用生物信息学分析方法进行了序列比较和进化树分析。结果重组质粒(pET28a-FLOTLLIN2)经双酶切及测序鉴定,证明插入片段序列与预期目的基因序列符合。SDS-聚丙烯酰胺凝胶电泳结果显示,体外原核表达的重组蛋白(rSjFLOTILLIN2)分子量大小为48 kD,其与预测的分子量大小一致。经Blast搜索比对发现,日本三角涡虫、白纹伊蚊、地中海果蝇、苜蓿切叶蜂等FLOTILLIN2序列与日本血吸虫FLOTILLIN2相似性最高;将日本血吸虫和热带爪蟾、原鸡等15种不同物种来源的FLOTILLIN2编码蛋白进行比较,并构建FLOTILLIN2系统进化树,系统发育分析结果显示,日本血吸虫与涡虫同为一支,遗传距离最为接近。结论日本血吸虫脂筏蛋白基因被成功克隆和表达,为进一步研究打下基础。     

T载体的构建及日本血吸虫肌动蛋白基因PCR产物的快速克隆

目的构建快速高效克隆PCR产物的克隆载体(T载体),并对日本血吸虫肌动蛋白全长编码基因PCR产物进行快速克隆.方法日本血吸虫肌蛋白全长编码基因的扩增采用反转录-聚合酶链反应(RT-PCR)方法.质粒pGEM5Zf ( )经限制性内切酶EcoR V 酶切,在仅含有脱氧胸苷三磷酸(dTTP)的PCR缓冲液中于70 ℃作用2 h,在每个片段的3'端加上一个脱氧胸苷(dT)碱基,构建成T载体.根据PCR扩增产物3'端存在一个非模板依赖的脱氧腺苷(dA)原理,将扩增产物直接克隆入T载体并测序.结果阳性克隆经琼脂糖凝胶电泳、限制性酶切分析、PCR及DNA序列测定等均证实克隆获得成功,且效率很高.与曼氏血吸虫肌动蛋白比较,核苷酸和推断的氨基酸的同源性分别是92.5%和99.7%.结论构建的pGEM5Zf-T载体对日本血吸虫肌动蛋白编码基因的PCR产物直接克隆既经济、简便,又快速、高效,所构建的T载体由于在插入位点两侧具有pUC/M13测序引物序列,可直接测定重组质粒中插入片段的核苷酸序列.所获得的日本血吸虫(大陆株)肌动蛋白编码基因与曼氏血吸虫有极高的同源性.     

日本血吸虫特异性IgE相关抗原编码基因的克隆和鉴定

目的　从日本血吸虫成虫cDNA文库中筛选并克隆日本血吸虫特异性IgE相关抗原编码基因。　方法　用ABC ELISA法从日本血吸虫病流行区筛选出高水平抗血吸虫成虫抗原IgE抗体的个体 15人 ,采集血清并混合。混合血清经Protein G柱吸收后 ,用于日本血吸虫成虫cDNA文库的免疫学筛选。PCR扩增阳性克隆插入cDNA片段。序列分析后 ,于该序列第一开读框两端设计引物并分别引入EcoRⅠ和NotⅠ位点 ,PCR扩增并纯化目的基因片段后克隆入质粒载体pGME T ,再亚克隆入表达载体pGEX 6p 1。经IPTG诱导表达 ,对表达产物进行SDS PAGE和Westernblotting鉴定。　结果　阳性克隆插入片段约 12 0 0bp ,第一开读框长 5 0 7bp ,编码 16 9个氨基酸 ,理论分子量为 19 3kDa。DNA序列分析显示 ,与已知序列同源性小于 4 0 %。重组质粒pGEX 6p 1/Sj4 3B能高效表达融合蛋白 ,且能被日本血吸虫特异性IgE抗体识别。　结论　成功构建的重组质粒pGEX 6p 1/Sj4 3B ,Sj4 3B可编码日本血吸虫特异性IgE抗体相关抗原     

日本血吸虫中国大陆株雌性特异性DNA片段及重复序列的分离与鉴定

目的分离鉴定日本血吸虫基因组DNA的雌性特异性片段及DNA重复序列.方法分别提取雌、雄日本血吸虫基因组DNA,制备检测子和驱动子;利用代表性差异分析(RDA)及PCR进行扣除杂交,获得目的DNA片段;对所获目的DNA片段用低熔点凝胶回收;转质粒载体、重组质粒载体的阳性克隆筛选、测序;用Southern blottmg对目的DNA片段进行鉴定.结果RDA及PCR获得5个目的DNA片段;并转质粒载体测定了5个目的DNA片段(P1～P5)的序列.Southernblottmg显示DNA片段P1和P2均与雌、雄基因组DNA强烈杂交;DNA片段P3与雌性基因组DNA的杂交强度明显高于与雄性基因组DNA的杂交强度;DNA片段P4和P5仅与雌性基因组DNA杂交,但不与雄性基因组DNA杂交.结论DNA片段P1和P2为日本血吸虫DNA重复序列,并以多拷贝存在于日本血吸虫基因组DNA中;DNA片段P3存在于日本血吸虫性染色体W和Z上,在性染色体W的拷贝数多于在性染色体Z上的拷贝数;DNA片段P4和P5仅存在于日本血吸虫性染色体W上,为日本血吸虫雌性特异性片段.     

日本血吸虫原肌球蛋白编码基因克隆和表达

目的　克隆和表达日本血吸虫大陆株原肌球蛋白 (tropomyosin ,TM)编码基因。方法　应用RT PCR方法体外扩增日本血吸虫原肌球蛋白编码基因 ,并采用T载体对该PCR产物直接进行克隆并用于核苷酸序列的测定。将目的基因亚克隆入原核表达载体pQE30 ,以IPTG诱导重组原肌球蛋白的表达。结果　PCR扩增产物约 82 3bp ,符合预计大小 ,并成功地克隆入T载体 ,对其中一克隆pGSjcTM12的插入片段的核苷酸序列分析表明 ,该序列和推测的氨基酸序列与曼氏血吸虫原肌球蛋白基因分别有 91 1%和 98 1%的同源性。该编码基因亚克隆入原核表达载体pQE30获得高效表达 ,分子量约 32kDa,并能被日本血吸虫天然原肌球蛋白免疫血清特异识别。结论　日本血吸虫原肌球蛋白编码基因克隆和原核表达成功。     

间日疟原虫云南分离株传播阻断疫苗候选抗原Pvs 28基因多态性分析

目的分析我国疟疾混合流行区云南分离株间日疟原虫（P．v）传播阻断疫苗候选抗原Pvs28基因特点。方法收集云南省血样17份；提取疟原虫基因组DNA；PCR扩增Pvs28基因；基因测序；DnaSP version4．0软件进行基因多态性分析。结果成功扩增Pvs28全长基因17个序列。与标准株Sal—I比较，检测出7个错义突变，7个基因型和7种氨基酸型。云南P.v分离株核苷酸多态性n值为0．0044。若不考虑重复片段拷贝数的差异，云南P．v分离株和湖北P．v分离株Pvs 28蛋白主导氨基酸型完全一致，即V^14-L^52-L^98-E^105-L^115-S^14-I^122。与泰国P．v分离株比较。我国主导基因型突变位点完全包含在泰国P．v分离株突变位点中。结论以Sal—I Pvs28为基础建立的传播阻断疫苗能够克服云南P．v分离株Pvs28抗原多样性而发挥传播阻断作用，提示间日疟原虫传播阻断疫苗在我国具有良好的应用前景。     

HIV-2 protease sequences of subtypes A and B harbor multiple mutations associated with protease inhibitor resistance in HIV-1

BACKGROUND: HIV-1 protease inhibitors (PI) have been used for treating HIV-2-infected persons but little is known about amino acid mutations associated with PI resistance in HIV-2 and whether they are similar to those seen in HIV-1. OBJECTIVE: To determine the frequency of HIV-1 PI resistance-associated mutations in PI-naive HIV-2-infected individuals. DESIGN: Using PCR, protease genes were amplified from 76 individuals, directly sequenced, phylogenetically subtyped, and translated into amino acids to analyze PI-associated major and minor mutations. RESULTS: Of the 76 HIV-2 sequences, 68% belonged to subtype A and 32% to subtype B. All sequences contained at least four codon changes giving substitutions at 10, 30, 32, 36, 46, 47, 71 or 77. The frequency of these mutations was similar in subtype A and B viruses. Two major resistance-conferring mutations, 30N and 46I, were identified in one (1%) and 68 (89%) specimens, respectively. Minor mutations 10V/I, 32I, 36I, 47V, and 71V were predominant (89%-100%), followed by the rare mutation 77I (1%). Of the 76 strains, 89% harbored multiple PI resistance-associated substitutions comprising both the major 46I and minor mutations: 10V/I, 32I, 36I, 46I, 47V, 71V (76%); 10V, 32I, 36I, 46I, 47V (9%); and 10V, 32I, 36I, 46I, 47V 71V, 77I (1.3%), 10V, 32I, 46I, 47V, 71V (1.3%), and 10V, 30N, 32I, 36I, 46I, 47V, 71V (1.3%). The remaining 11% of the sequences had patterns with only minor mutations: 10V, 32I, 36I, 47V, 71V (9%) and 10V, 32I, 36I, 47V (1.3%). CONCLUSIONS: The high frequency of multiple PI-associated substitutions represent natural polymorphisms occurring in HIV-2 strains of subtypes A and B. Phenotypic and clinical studies are needed to determine the relevance of these substitutions.     

我国间日疟原虫传播阻断疫苗候选抗原Pvs48基因特点分析

目的明确我国疟疾流行区间日疟原虫传播阻断疫苗候选抗原Pvs48基因特点。方法收集疟疾单纯流行区浙江、湖北两省间日疟患者血样,制备血样干滤纸片;提取疟原虫基因组DNA;聚合酶链反应(PCR)扩增Pvs48基因;Sanger双脱氧链终止法测序并分析。结果成功扩增12例间日疟原虫分离株Pvs48全长基因。与间日疟原虫标准株SalⅠ比较,检测出6个错义突变位点,导致6处氨基酸置换和6种基因型。结论我国间日疟原虫传播阻断疫苗候选抗原Pvs48具有保守性。     

Molecular Genetic Analysis of the ABO Blood Group System: 3. Ax and B(A) Alleles

We have employed a PCR approach to determine the nucleotide sequences of the coding region in the last two coding exons of the histo-blood group ABO genes from one A_x and one B(A) individual. Compared with A¹ alleles, the A_x allele has a single nucleotide substitution (T→ A at nucleotide 646) resulting in an amino acid substitution (phenylalanine→isoleucine at amino acid 216). Compared with B alleles, the B^(A) allele has two nucleotide substitutions (T→C at nt. 657 and A→G at nt. 703) resulting in an amino acid substitution (serine→glycine at aa. 235). The amino acid substitution resulting from this B^(A) allele is located at the second of the four amino acid substitutions which discriminate human A and B transferases, and the amino acid residue (glycine) is identical to that of A transferase suggesting the involvement of this amino acid or its surrounding area for the recognition and/or binding of the donor nucleotide sugars.     

日本血吸虫组织蛋白酶L1基因的编码区全序列分析及克隆

目的 分析日本血吸虫组织蛋白酶L1(SjCL1)基因编码区的完整序列,并定向克隆到真核表达质粒pcD-NA3中。 方法 从日本血吸虫成虫提取总RNA,进行反向巢式RT-PCR,T载体克隆后测序。PCR扩增SjCL1基因的编码区序列,并将扩增产物克隆到pcDNA3质粒的BamHI和Xhol位点上。结果 通过反向巢式RT-PCR扩增出332 bp SjCL1基因5’端序列,测序后与报道的SiCL1基因部分序列拼接,可得到一个编码317个氨基酸的完整编码区序列。PCR特异性扩增出SjCL1编码区基因序列,其大小约为1 kb。经酶切、PCR鉴定和测序表明所构建的质粒pcDNA-SjCL1中含有所扩增的基因序列。 结论 构建了含SjCL1基因的编码区序列的真核表达质粒pcDNA-SjCL1。     

Polymerase chain reaction (PCR) mutagenesis enabling rapid non-radioactive detection of common β-thalassaemia mutations in Mediterraneans

The IVS-1-110 (G----A) and IVS-1-1 (G----A) mutations occur in approximately 33% and 9% respectively of beta-thalassaemia alleles in Mediterraneans (Kazazian & Boehm, 1988). They are generally detected in polymerase chain reaction (PCR)-amplified material by allele-specific oligonucleotide (ASO) hybridization patterns. In this study, artificial base substitutions in amplified material have been created to distinguish normal from mutant alleles on the basis of restriction enzyme digestion patterns. Invariant target sites provide an internal control for restriction enzyme activity. Mutagenesis was achieved by 3' base mismatches in primers selected to anneal immediately adjacent to target sites. Digestion of PCR products from normal and thalassaemic alleles with the restriction enzymes MboI (IVS-1-110) and HinfI (IVS-1-1) produced different fragments on electrophoresis. The above strategy was validated by allele-specific oligonucleotide probing. Identification of the three commonest mutations in this population (IVS-1-110, codon 39 and IVS-1-1), which account for approximately 69% of thalassaemic alleles (Kazazian & Boehm, 1988), was subsequently undertaken in seven chorion villus biopsies.     

An HphI-polymorphism in exon 28 of the von Willebrand factor gene, and its frequency among patients with various forms of von Willebrand''s disease

Besides having a large number of restriction fragment length polymorphisms (RFLP) the von Willebrand factor (vWF) gene contains several sequence polymorphisms in the coding regions. Eight nucleotide substitutions have been reported in two or more independent cDNA clones. Four of them give rise to amino acid substitutions, two of which are in the mature vWF subunit (at positions 26 and 709). We have investigated a previously suggested putative alanine-threonine polymorphism at position 618 of the mature subunit in normal subjects and patients with various types of von Willebrand's disease (vWD). the codon for amino acid 618 is located in exon 28, which encodes several important vWF functional domains. We amplified the whole exon 28 and parts of it by polymerase chain reaction (PCR) and distinguished gene from pseudogene sequences. The alanine----threonine (G----A) substitution was studied with restriction enzyme cleavage of the products, since it creates a new HphI site. Moreover, in two individuals we confirmed the polymorphism by cDNA sequencing. In 23 normals the frequencies of the h- (Ala) and the h+ (Thr) alleles were 0.50/0.50. In eight patients with type III vWD from seven different families, the h- allele was present in 13 of 16 genes, but whether this signifies a common mutation in some of the patients is not known. In types I and II, both alleles were present in roughly similar proportions. Owing to the high frequency of heterozygosity, the polymorphism should prove useful as an aid in genetic counselling.     

Conserved sequences of thrombospondin-related adhesive protein gene of Plasmodium vivax in clinical isolates from Korea

Thrombospondin-related adhesive protein (TRAP) from Plasmodium vivax (P. vivax) became one of the important vaccine candidates for malaria, because P. vivax TRAP (PvTRAP) is responsible for the sporozoite-host interactions. PvTRAP polymorphisms in the isolates from Republic of Korea (ROK) were analysed, setting the valuable baseline data for the future vaccine developments and clinical trials with PvTRAP, as a strong vaccine candidate. A total of 54 isolates were collected in 2010. PvTRAP genes from above isolates were amplified and sequenced, and the results were analysed and compared against Sal-1 strain. Sequencing analysis of 1424-bp-size PvTRAP PCR products revealed one major allelic type with six non-synonymous substitutions, where S81T, E95D, I121V and T127R substitutions were found in region II, and K371N and A425E substitutions from region IV. The ROK isolates revealed the limited sequence polymorphisms in PvTRAP in comparison with the reported isolates from other nations.     

日本血吸虫组织蛋白酶L2基因扩增及克隆

目的　体外扩增日本血吸虫中国大陆株组织蛋白酶L2 (SjCL2 )的编码区基因序列 ,将其克隆到真核表达质粒pcDNA3中 ,为进一步对其进行功能研究奠定基础。方法　用TRIZOL分离日本血吸虫成虫RNA ,RT -PCR扩增目的基因 ,将扩增产物定向克隆到真核表达载体 pcDNA3中。 结果　RT -PCR特异性扩增出SjCL2编码区基因序列 ,其片段大小为1kb左右 ,经酶切和PCR鉴定表明所构建的质粒pcDNA -SjCL2中含有所扩增的基因序列。 结论　RT -PCR扩增的SjCL2编码区基因序列与预期长度相符合 ,成功构建了含SjCL2编码区基因的真核表达质粒pcDNA -SjCL2。     

Haemophilia A mutations in the UK: results of screening one-third of the population

One-third of the UK haemophilia A population was screened to establish a national database of mutations and pedigrees and advance knowledge of the disease. The following mutations were found: 131 intron 22- and 13 intron1-breaking inversions; 11 gross deletions and an insertion; 65 frameshifts; three in-frame deletions and one insertion; 46 nonsense; 30 intronic mutations affecting splice sites and four generating new sites; 469 non-synonymous mutations due to 203 different base substitutions of which four affected, and nine were predicted to affect, splicing; three promoter mutations; two synonymous exon 14 mutations possibly affecting splicing; two VWF mutations. Of the above mutations, 176 are not listed in the Haemophilia A Mutation, Structure, Test and Resource Site (HAMSTeRS). Four gross deletions arose by non-homologous end-joining; we detected unexpected splicing in some mutations; substitution of amino acids conserved for less than 90 million years are rare; the risk of developing inhibitors for patients with nonsense mutations is greater when the stop codon is in the 3' half of the mRNA; changes likely to generate splice sites causing frameshifts are over-represented among non-synonymous mutations associated with inhibitors; our data and those in HAMSTeRS enabled the size of the spectrum of specific mutations causing the disease to be estimated and to determine how much of it is known.