RT-PCR构建人甲胎蛋白全长基因真核表达载体

目的 采用反转录PCR(RT-PCR)构建人甲胎蛋白 (Alpha-Fetoprotein, AFP)全长基因真核表达载体,为下一步应用以AFP蛋白为靶点的基因治疗作准备。方法 体外培养人肝癌细胞HepG2,Trizol裂解细胞,提取总mRNA。设计含有特定酶切位点BglⅡ,SalⅠ和真核启动元件Kozak序列的RT-PCT正反向引物,采用RT-PCR法克隆AFP全长cDNA,使用BglⅡ +SalⅠ双酶切载体pEGFP-C3载体和AFPcDNA,将AFPcDNA连接载体pEGFP-C3,构建新的真核表达载体,命名为pEGFP-AFP。结果 通过测序证明pEGFP-AFP真核表达载体含有AFP全长基因。结论:本研究成功构建人甲胎蛋白基因真核表达载体pEGFP-AFP,为进一步的分子试验和基因治疗打下了基础。

人FHIT基因实时荧光定量RT-PCR检测方法的建立

目的:建立检测人脆性组氨酸三联体(fragile histidine triad,FHIT)基因的荧光定量RT-PCR方法。方法:根据GenBank中人FHIT基因序列,在保守区设计合成一对特异引物,将PCR扩增得到的FHIT基因克隆至PMD18-T载体上,构建的重组质粒标准品倍比稀释后运用SYBR Green I染料法绘制标准曲线,并进行融解曲线分析。结果:FHIT基因的标准品稀释度在1×107～1×103copies/μl范围内具有良好的线性关系,Ct值线性范围为14.28～28.36,相关系数为0.998,融解曲线分析表明,产物为特异的单峰,Tm为92.3±0.1℃。结论:建立了人FHIT基因实时荧光定量RT-PCR方法,为在mRNA水平对人FHIT的定量分析奠定了基础。

Designing Primers for H5 and H7 Subtypes of Avian Influenza Virus and Multiplex RT-PCR Amplification

[Objective] The research aimed to design primers that are suitable for detecting H5 and H7 subtypes of avian influenza virus （AIV） ; [Method] DNAStar was used to analyze the homology of the sequences of H5 and H7 subtypes of AIV accessed in GenBank, and design primers（ by Primer Premier 5.0） on high homologous region of these sequences, and then amplified by RT-PCR. [Result] The multiplex RT-PCR amplification, agarose gel electrophoresis and sequencing results showed that the self-designed primers are successful for detecting AIV. [Conclusion] It is feasible to rapidly diagnose AIV through this method.

Selection of reference genes for qRT-PCR analysis of gene expression in sea cucumber Apostichopus japonicus during aestivation

Quantitative real-time reverse transcription-polymerase chain reaction （qRT-PCR） is a technique that is widely used for gene expression analysis, and its accuracy depends on the expression stability of the internal reference genes used as normalization factors. However, many applications of qRT-PCR used housekeeping genes as internal controls without validation. In this study, the expression stability of eight candidate reference genes in three tissues （intestine, respiratory tree, and muscle） of the sea cucumber Apostichopus japonicus was assessed during normal growth and aestivation using the geNorm, NormFinder, delta CT, and RefFinder algorithms. The results indicate that the reference genes exhibited significantly different expression patterns among the three tissues during aestivation. In general, the β-tubulin （TUBB） gene was relatively stable in the intestine and respiratory tree tissues. The optimal reference gene combination for intestine was 40S ribosomal protein S18 （RPSI 8）, TUBB, and NADH dehydrogenase （NADH）; for respiratory tree, it was β-actin （ACTB）, TUBB, and succinate dehydrogenase cytochrome B small subunit （SDHC）; and for muscle it was α-tubulin （TUBA） and NADH dehydrogenase [ubiquinone] 1α subcomplex subunit 13 （NDUFA13）. These combinations of internal control genes should be considered for use in further studies of gene expression in A.japonicus during aestivation.

Detection of Survivin mRNA in nasopharyngeal carcinoma by real-time fluorescence quantitative RT-PCR

Objective： To establish the method of real time fluorescence quantitative RT-PCR for detecting the expression of Survivin mRNA in nasopharyngeat carcinoma （NPC） tissues. Methods： The total RNA was extracted from NPC cell line CNE-2 and tissues with Trizol and then been transcribed reversely to cDNA, a method of real time fluorescence quantitative RT-PCR for detecting the expression of Survivin mRNA in NPC tissues had been established, in which chronic nasopharyn-gitis patients＇ nasopharynx tissues treated as control group. Results： The expression of Survivin mRNA all could be detected either in CNE-2 cells, NPC tissues or in chronic nasopharyngitis patients＇ nasopharynx tissues, and there was higher the expression level of Survivin mRNA in NPC tissues than which in chronic nasopharyngitis patients＇ nasopharynx tissues, the difference was significant （P 〈 0.01）. The expression of Survivin mRNA could be detected both in stage Ⅰ ＋ Ⅱ and stage Ⅲ ＋ Ⅳ NPC, and there was no significant difference in relative quantifications of gene expression between these two groups （P 〉 0.05）. There was no relationship between Survivin mRNA expression and age and sex of NPC patients （P 〉 0.05）. Conclusion： Real time fluorescence quantitative RT-PCR is a rapid, effective and high sensitive method for detecting the expression of Survivin mRNA in NPC tissues. The overexpression of Survivin mRNA may play some roles in pathogenesis of NPC.

Quantification of Simian Immunodeficiency Virus by SYBR Green RT-PCR Technique

Plasma viral RNA load is widely accepted as the most relevant parameter to assess the status and progression of Simian immunodeficiency virus （SIV） infections. To accurately measure RNA levels of the virus, a one-step fluorescent quantitative assay was established based on the SYBR green Real-time reverse transcription-polymerase chain reaction （RT-PCR）. The lower detection limit of the assay was 10 copies per reaction for the virus. This method was successfully applied to quantify SIVmac251 and SIVmac239 viruses produced in CEMx174 cells. Additionally, the performance of the SYBR green RT-PCR was assessed in a SIVmac251 infected rhesus macaque. The result demonstrated that the method could detect as little as 215 copies per milliliter of plasma and the dynamic pattern of viral load was highly consistent with previous results. With regard to convenience, sensitivity and accuracy our assay represents a realistic alternative to both branched-chain DNA （b-DNA） assays or real-time PCR assays based on TaqMan probes.

Establishment of an orthotopic transplantation tumor model of hepatocellular carcinoma in mice

AIM:To improve the outcome of orthotopic transplantation in a mouse model,we used an absorbable gelatin sponge(AGS) in nude mice to establish an orthotopic implantation tumor model.METHODS:MHCC-97L hepatocellular carcinoma(HCC)cells stably expressing the luciferase gene were injected into the subcutaneous region of nude mice.One week later,the ectopic tumors were harvested and transplanted into the left liver lobe of nude mice.The AGS was used to establish the nude mouse orthotopic implantation tumor model.The tumor suppressor gene,paired box gene 5(PAX5),which is a tumor suppressor in HCC,was transfected into HCC cells to validate the model.Tumor growth was measured by bioluminescence imaging technology.Semi-quantitative reverse transcription polymerase chain reaction(RT-PCR) and histopathology were used to confirm the tumorigenicity of the implanted tumor from the MHCC-97L cell line.RESULTS:We successfully developed an orthotopic transplantation tumor model in nude mice with the use of an AGS.The success rate of tumor transplantation was improved from 60% in the control group to 100% in the experimental group using AGS.The detection of fluorescent signals showed that tumors grew in all live nude mice.The mice were divided into 3 groups:AGS-,AGS+/PAX5-and AGS+/PAX5 +.Tumor size was significantly smaller in PAX5 transfected nude mice compared to control mice(P < 0.0001).These fluorescent signal results were consistent with observations made during surgery.Pathologic examination further confirmed that the tissues from the ectopic tumor were HCC.Results from RT-PCR proved that the HCC originated from MHCC-97L cells.CONCLUSION:Using an AGS is a convenient and efficient way of establishing an indirect orthotopic liver transplantation tumor model with a high success rate.

SEQUENCE ANALYSIS OF THE NS5 REGION OF GBVC/HGV AND DETECTION OF THE VIRUS BY REVERSE TRANSCRIPTASE PCR

GBV C/HGV is a newly identified virus associated with human hepatitis In this study, the nucleotide sequences of the partial NS5 gene of GBV C/HGV derived from sera of 8 Chinese patients were determined The nucleotide homology among the 8 isolates were 92% on average On the basis of sequence analysis, two sets of oligonucleotide primers derived from highly conserved region of GBV C/HGV NS5 gene were designed to establish both sensitive and specific nested PCR for detection of GBV C/HGV RNA 253 Chinese patients were examined for the virus RNA GBV C/HGV RNA positive rates in patients infected with HBV, HCV and patients with chronic non B,non C hepatitis were 18 4%, 19 8% and 8 9% respectively This result suggested that HBV,HCV and GBV C/HGV shared the same transmission risk factors 8 patients with GBV C/HGV and HCV coinfection were retrospectively observed for the response to interferon Coinfection with GBV/HGV did not negatively influence the responsiveness of HCV, and GBV C/HGV was sensitive to interferon to a certain degree

PARTIAL DELTETION OF p53 GENE IN α PARTICLE－INDUCED TRANSFORMANT OF SYRIAN HAMSTER EMBRYO CELLS

The mutation of p53 gene was detected in Syrian hamster embryo(SHE) cells neoplastically initiated with a particles.The level of the p53 mRNA in transformant was obviously higher than that in non-irradiated counterpart,as measured by Northern blot analysis of total RNA.A pair of primers were designed based on p53 cDNA sequence to produce the whole length of coding sequence about 1.2 kilobase(Kb) by reverse transcription of mRNA followed by the polymerase chain reaction(RTPCR),but the length of fragment amplified from transformant mRNA was about 0.3Kb,remarkably shorter than that from normal SHE cells.Imrnunohistochemical analysis of p53 protein showed that no heavy staining was found on slice of tumor derived from transformant inoculated in nude mice with hamster specific p53 monocloned antibody HD200.The results implied that p53 gene had been mutated by deletion,which might lead to loss of p53 protein expression but the increased expression of p53 remained in a particle-induced SHE transformant.

Optimization of Quantitative Real-time PCR System on Amplification of Beta-glucosidase Gene Os1bglu4

[Objective] This study aimed to establish a quantitative real-time PCR （qRT-PCR） system for detecting the expression of rice beta-glucosidase gene Os1bglu4.[Method] The PCR was conducted with SYBR Green Ⅰ method,using the primers of reference gene actin or ubiquitin.[Result] Actin was more suitable to be the reference gene than ubiquitin.More accurate results were obtained when the 100 ng cDNA template was added at a large volume and a lower concentration.The primer concentration in the range from 0.2 to 0.8 μmol/L we set had no significant influence on the results,so,0.4 μmol/L was selected as the optimal primer concentration in this study.The amplification efficiency was greatly reduced when the annealing temperature was set at 64 ℃,therefore,annealing temperature was set at 60 ℃.Compared with the reaction system of 25 μl,the fluorescence intensity was significantly lower but the CT value did not change greatly in 10 μl system.So,the 10 μl reaction system was selected,which significantly reduces the research costs for the detection of a large amount of samples in future study.

Cloning of cDNA Encoding COMT from Chinese White Poplar ( Populus tomentosa ), Sequence Analysis and Specific Expression

The cDNA fragment encoding caffeic acid 3_O_methyltransferase (COMT) in Chinese white poplar ( Populus tomentosa Carr.) was isolated and cloned by RT_PCR technique. The size of the cDNA fragment is 1 080 bp, which almost covers the whole cDNA_encoding region. Authors’ cDNA fragment in P. tomentosa shares 98.7% homology with the reported corresponding cDNA in the P. tremuloids at nucleotide level, 99.4% homology at amino acid level, respectively. The analysis of Northern dot hybridization showed that COMT is expressed specifically in the developing secondary xylem of stem during the season of xylem differentiation, which means the linkage between the gene expression for a monolignol biosynthetic enzyme and seasonal regulation of xylem development in woody plant.

Developmental Changes of Col3a1 mRNA Expression in Muscle and Their Association with Intramuscular Collagen in Pigs

Eighty-four castrated boars including Laiwu Black （LW） （weight 30-90 kg, n = 6） and Lulai Black （LL） （weight 40-100 kg, n = 6） were used to study the developmental changes of collagen type Ⅲ alpha 1 （Col3al） mRNA expression in the muscle and their association with intramuscular collagen （IMC）. The muscle total RNA was extracted to determine the abundance of Col3al mRNA using relative quantitative RT-PCR with β-actin mRNA as the internal standard. The results indicated that the developmental patterns of muscle Col3al mRNA in LW and LL pigs were similar. The abundance of Col3al mRNA increased with body weight, but decreased a little at 70 kg and 80 kg phases for LW and LL, respectively. On the whole, the expression level of Col3al mRNA in muscle of LW was higher than that of LL （P 〈 0.05）. Correlation analysis showed that the expression of Col3al mRNA in muscle was positively correlated with total and insoluble IMC, but was negatively correlated with IMC solubility for LW pigs （P 〈 0.01） and LL pigs （P 〈 0.05）, respectively. These results suggest that the muscle Col3al gene expression is affected by body weight and genotype and has important effect on IMC content and characteristics.

Cloning and Expression of a Profilin Gene from Rapeseed

Profilin has recently been identified as an actin-binding protein in higher plants. A cDNA clone (designated Repro) encoding profilin gene was isolated from rapeseed ( Brassica napus L. cv. canadian Tween) using RT-PCR technique. Sequence analysis showed 82% similarity to Zea mays L. ZmPro3, 85% to Arabidopsis AthPRF1, 82% to Nicotiana tabacum L. NTPRO, 81% to Oryza sativa L. profilin A. A new full-length cDNA was obtained by 5'-RACE and 3'-RACE techniques. Sequence analysis showed that the size of full-length cDNA is 672 bp which contains a major open reading frame of 134 amino, acids, 5' and 3' untranslated regions and a long Poly (A) tail. Northern blot analysis showed that the profilin gene is a pollen and anther specific gene.

Cloning and Analysis of ISA1 from Oryza sativa

[Objective] The aim was to clone ISA1 from Oryza sativa and analyze its expression situation in different tissues and different endosperm filling stage.[Method] With japonica rice cultivar nipponbare as test material,the expression patterns of ISA1 in different tissues and different endosperm filling stage was analyzed by using semi-quantitative RT-PCR technique.[Result]The full length open reading fragments of ISA1 encoded 811 amino acid residues.The homologous alignment and phylogenetic analysis showed th...

Detection of the SYT-SSX Fusion Gene in Synovial Sarcoma

Objective: To investigate the feasibility and significance of detecting SYT-SSX fusion gene in paraffin-embedded tissues of synovial sarcoma (SS) by reverse-transcriptase polymerase chain reaction(RT-PCR) methods. Methods: Twenty cases of SS tumors from archival materials were collected and all samples were formalin-fixed and paraffin-embedded (FFPE). SYT-SSX fusion transcript was detected by RT-PCR. Home-keeping gene Porphobilinogen Deaminase (PBGD) was regarded as internal control.Results: PBGD mRNA was detected in all 20 tumor cases (100%). SYT-SSX fusion transcript was detected in 18 tumor cases (90%). In 18 SYT-SSX positive SS cases, there are 12 present SYT-SSX1 fusion transcript and 6 present SYT-SSX2 fusion transcript. SYT-SSX1 fusion transcript can be seen in 9 monophasic SS and 3 biphasic SS. In 6 SYT-SSX2 positive SS cases, 4 were monophasic SS and 2 were biphasic. Conclusion: Detection of SYT-SSX fusion transcripts in FFPE tissues for diagnosis of SS is feasible and sensitive. Subtypes of SYT-SSX fusion gene may provide prognosis information.

cDNA Cloning and Expression Analysis of Mest Gene in the Bufo gargarizans

The Mest （mesoderm-specific transcript） gene has been considered an imprinting gene in human and mouse, and was also confirmed in other mammals and flowering plants. To investigate the function and evolution of this gene, the cDNA of full length Mest gene was obtained using 5＇- and 3＇-RACE from the Chinese Large Toad （Bufo gargarizans）. The transcript is 1 325bp in length which contains a complete open reading frame （ORF） encoding a polypeptide of 326 amino acids （GenBank accession number： ABQ10905）. There is a typical 0./13 hydrolase fold domain in the putative gene product, and it shows high similarity to sequence of homologous protein of Xenopus tropicali （86%）, mammlian （70% - 80%）. RT-PCR （reverse transcriptase-polymerase chain reaction） analysis demonstrated that the Bufo gargarizans Mest （BgMest） gene is expressed widely in testis, ovary, liver, kidney, spleen, brain, stomach and lung. The conservation of the BgMest gene sequences, protein secondary structure of the BgMest protein, in addition to the expression pattern of the BgMest gene, suggested that the function of BgMest was conserved in amphibians. However, the phylogenetic tree of the imprinting gene of the mammals and other vertebrates examined in this study indicated their divergent origins.

Cloning and Analysis of the cDNA Sequence of XTH Gene from Chrysanthemum morifolium Petal

[Objective] This study aimed to investigate the structural character of xy- Ioglucan endotransglycosylase/hydrolase （XTH） gene in ethylene-insensitive feverfew. [Method] The total RNA was extracted from Chrysanthemum rnorifolium petal using Trizol reagent, and the cDNA fragment of XTH gene was cloned by RT-PCR and T/A cloning. [Result] The sequencing result showed that the cloned cDNA sequence was 911 bp. It was predicted to encode a polypeptide of 293 amino acids and had seven active sites of XTH family, and then named as CmXTH （gene accession number HM752243）. In addition, The BLAST analysis showed that the deduced amino acid sequence of CmXTH showed high homology with other 19 chosen plant XTHs. Among of these, CmXTH shared closer genetic relationship with Gerbera jamesonii, Solanum lycopersicum, whereas had relatively distant relationship with Populus euparatica, Fragaria ananassa, Actinidia deliciosa, etc. [Conclusion] The cloned fragment was certainly the cDNA of XTH gene, which was associated with the petal growth and senescence in Chrysanthemum morifolium.

Study on the Gene Transcription Level of Hippocampus NGF in C_(57) Mouse Development

β-Nerve growth factor (β NGF) mRNA levels in hippocampa from C57BL/6J mice of different ages were compared by semi-quantitative RT-PCR method, taking β-actin gene as an internal control. The levels of 6-and 12-month-old C57 mice were much lower compared with that of the l-month-old mouse. However, there was no significant difference between these two groups. This result suggests that gene transcription level of hippocampus β NGF in a C57, mouse descends evidently as it grows up. Base T at Site 294 of β NGF cDNA was substituted by C in the C57, mouse, as revealed by DNA sequencing for the first time. Nevertheless, this polymorphism of nucleotide did not make any difference in amino acid composition.

Plasmolysis treatment enhances the expression of callose synthase gene in zygotic embryos of Eleutherococcus senticosus

In previous study we reported that pretreatment with plasmolysis enhanced somatic embryo formation in hypocotyls of Eleutherococcus senticosus.In the present study,the expression level of callose synthase gene in embryos of E.senticosus in response to 2,4-D,sucrose and mannitol treatments was analyzed by RT-PCR.The results show that plasmolysis pretreatment using sucrose and mannitol significantly promoted the expression of callose synthase gene.Also,the thicker cell walls of explant plasmolyzed compared with controls were observed during the somatic embryogenesis.We suggest that the callose may make the cells in epidermis separate from neighboring cells and then develop into embryogenic potential cells.

Human papilloma virus 16 E6 oncoprotein associated with p53 inactivation in colorectal cancer

AIM:To investigate the association between human papilloma virus(HPV) infection and colorectal cancer.METHODS:Sixty-nine patients with pathologically confirmed primary colorectal cancer including 6 stage Ⅰ,24 stage Ⅱ,21 stage Ⅲ,and 18 stage Ⅳ patients were enrolled in this study to investigate whether HPV 16 could be involved in colorectal tumorigenesis.Nested-polymerase chain reaction(nested-PCR) was used to detect HPV16 DNA in colorectal tumor tissues and further confirmed by in situ hybridization(ISH).In addition,immunohistochemistry analysis was performed to examine the E6 oncoprotein in colorectal tumors.To verify whether E6 could inactivate the p53 transcriptional function,the levels of p21 and Mdm2 mRNA expression were evaluated by real-time reverse transcription(RT)-PCR.RESULTS:Of the 69 colorectal tumors,HPV16 DNA was detected in 11(16%) by nested-PCR,and HPV16 DNA was present in 8 of the 11(73%) tumors which was confirmed by ISH.The presence of HPV16 DNA in colorectal tumors was not associated with patients' clinical parameters including age,gender,smoking status,tumor site;however,HPV16 infection was more common in stage Ⅰ patients than in late-stages patients(Ⅱ,Ⅲ and Ⅳ).We next asked whether HPV16 infection could be linked with colorectal cancer development.Immunohistochemical data indicated that 8 of the 11 HPV16 DNA-positive tumors had E6 oncoprotein expression.Moreover,we also observed that the adjacent normal tissues including endothelial cells,lymphocytes,fibroblasts,and gland cells in E6-positive tumors had E6 oncoprotein expression.In addition,3 of the 4(75%) E6-positive tumors carrying p53 wild-type had negative immunostaining,but one tumor had less p53 immunostaining.We further examined whether E6-positive and/or p53 mutated tumors reduce p53 transcriptional activity.Real-time RT-PCR analysis indicated that p21 and mdm2 mRNA expression levels in E6/p53-wildtype tumors were significantly lower than in their adjacent normal tissues;as expected,E6-positive/p53-mutated tumors had lower p21 and mdm2 mRNA expression levels compared with their adjacent normal tissues.These results clearly indicate that the E6 oncoprotein expressed in p53 wildtype tumors may reduce p21 and mdm2 expression via p53 inactivation.CONCLUSION:These results suggest that HPV16 infection may be involved in a subset of colorectal cancer,and we suggest that the transmission of HPV to the colon and rectum might occur through peripheral blood lymphocytes.