结核分枝杆菌MPT64蛋白的表达、纯化及初步应用

目的 获得重组MPT64蛋白 ,研究其免疫学特性 ,评价其在结核病血清学诊断中的价值。方法 应用基因工程技术表达、纯化MPT64蛋白 ,通过Westernblotting分析其抗原性。分别以结核分枝杆菌PPD或纯化的rMPT64蛋白为抗原 ,通过ELISA方法及结核病一步法检测血清中抗结核抗体。结果 重组质粒pET1 5b MPT64测序表达除 83位密码子由CCA突变为CCG外 ,其余密码子均与报道的相同 ,但其氨基酸序列无变化。它在大肠杆菌BL2 1 (DE3)细胞内以包涵体形式存在 ,表达量占菌体总蛋白的 2 0～ 30 % ,分子量约 2 6kDa ,Westernblotting分析它与抗结核分枝杆菌多克隆抗体具有良好的免疫反应性。纯化后的rMPT64样品经SDS PAGE和光密度扫描分析表明其纯度约为 80 %左右 ,每1 0 0ml培养菌可获得 1 0mg左右的重组蛋白。以 33例正常人血清的OD值 + 2S为正常界限值 ,PDD的特异性和敏感性分别为 94% ( 31 /33)、63.7% ( 2 1 /33)、66.7% ;rMPT64纯化蛋白分别为 94% ( 31 /33)、30 .3( 1 0 /33) ;一步法分别为 88% ( 2 9/33)、66.7% ( 2 2 /2 3)。结论 pET1 5b MPT64大肠杆菌工程菌能以包涵体形式高效表达重组MPT64蛋白 ,该蛋白具有较高的抗原特异性和免疫反应性。rMPT64纯化蛋白有可能成为结核病血清学诊断的组合抗原之一。

Transformation of Pea Lectin Gene and Parasponia Haemoglobin Gene into Rice and Their Expressions

Lectin and leghemoglobin in legumes play the important roles, respectively, in recognition of host plants to their rhizobial bacteria, and lowering the oxygen partial pressure around bacteroids and protecting nitrogenase from oxygen in symbiotic nitrogen-fixing nodules. In order to extend the host range of the rhizobial bacteria and to make them fix nitrogen in non-legumes, pea lectin gene (pl) and Parasponia hemoglobin gene ( phl,) have been constructed into a plant expression vector (pCBHUL) and the vector pCBHUL was introduced into rice calli from immature young embryos by particle bombardment. After the calli were regenerated into plantlets on the resistant-selecting media containing hygromycin, they were identified by PCR and Southern blot hybridization. It was indicated that the pi and phb genes were integrated into nucleic genome of the transformed rice plants. GUS activity and the product of the pi gene were determined by GUS staining, Western blot and in situ hybridization at translational level. Eighteen out of 40 plants resistant to hygromycin were positively identified by PCR analysis with the rate of 45%. The pi gene was expressed in 3 out of 18 plants with 17% and 7.5% in 40 plants. The results may provide a clue for exploring whether Rhizobium leguminosarum by. viceae could extend its host range and make the transgenic rice plants have the possibility of being symbiotic, or associative to nitrogen fixation.

Cloning and Analysis of CFL—A LFY_like Gene from Cucumber

LFY and LFY _like genes have been shown to control the initiation of floral meristems in higher plants. The homologous cDNA of LFY, CFL, were cloned from cucumber ( Cucumis sativus L.). Southern blot analysis showed it was a single copy gene in the cucumber genome. Northern blot analysis showed that it expressed in the floral buds and young leaves. The possible role of CFL in the floral and vegetative development of cucumber plant was discussed.

Selection of Reference Genes in Transcription Analysis of Gene Expression of the Mandarin Fish, Siniperca chuasti

At present, transcription analysis of gene expression commonly uses housekeeping genes as control for normalization. In this study, the expression levels of three housekeeping genes including GAPDH, β-actin, and 18S rRNA in six tissues and five developmental stages of the Mandarin fish Siniperca chuatsi were assayed with quantitative real-time PCR （qPCR）. Differences in expression levels were analyzed using geNorm program. The results demonstrate that β-actin is the most stable gene at developmental stages and GAPDH is the most stable in different tissues. While 18S rRNA expression during development is differentially regulated, which indicates it is suitable as an internal control for gene expression normalization at the developmental level. Overall, the data suggest that the two most stable housekeeping genes are enough to accurately calibrate gene expression in S. chuatsi. The significance of this study provided convincing references and methodology for housekeeping gene selection and normalization in gene expression analysis with regular PCR or qPCR.

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.

Isolation, Expression Characteristics and Chromosomal Locations of Three cDNA Fragments Under Salt Stress in Rice

cDNA libraries were constructed from the leaves of a rice (Oryza sativa L.) salt tolerancevariety Tesan抋i 2 growing in solutions with 150 mmol/L NaCl for 3 h or without salt stress. Three salt-responsive cDNA clones, Ts1, Ts2 and Ts3 were isolated by differential screening. Northern blottinganalysis showed that the transcription levels of Ts1 and Ts2 increased within 3 h salt stress and kept onincreasing within 24 h, while the transcription level of Ts3 reached its peak within 3 h. Sequence analysisindicated that there were no homologies between the three cDNA clones and any known gene. The threecDNA clones were mapped using a doubled haploid (DH) population derived from an indica variety ZYQ8,which was a salt tolerance parent of Tesan抋i 2, with a japonica variety JX17. Ts1, Ts2 and Ts3 werelocated on chromosomes 1, 3 and 7, respectively. It was noted that Ts1, Ts2, and Ts3 were in or near theregions of major or minor salt tolerance quantitative trait loci (QTLs), which were mapped in the same DHpopulation in a parallel study.

Related Study on the Nm23-H1 Gene Expression in the Model of Ovarian Carcinoma Cell Lines with High Frequent Metastasis

Objective: To select the ovarian carcinoma cell lines with high frequent metastasis and study the association between nm23-H1 gene expression and metastasis of ovarian carcinoma. Methods: Each ovarian cancer cell line was transplanted subcutaneously into the flank of nude mice, and the metastatic behavior was evaluated by counting lung tumor foci at different time points. The metastatic tumors were cultured in vitro, then substrain was established and transplanted subcutaneously three times. The RNA level of nm23 in 8 human ovarian cancer cell lines were examined by northern-blot. Results: Of the 8 human ovarian cancer cell lines, 4 had high requent metastatic potentiality. The expression of nm23 RNA in human ovarian cancer cells was inversely related to metastatic behavior in the experimental animals (r=0.96, P=0.0001). Conclusion: The difference of the tendency of metastasis which was determined by genetic and molecular levels was significant among different type of cell lines and subtypes. The expression of nm23 mRNA in human ovarian carcinomas was correlated closely with the reduced metastatic behavior in experimental animals and may serve as a sensitive prognostic indicator for ovarian cancer.

Cloning and Differential Expression of a 1-Aminocyclopropane-1-Carboxylate Synthase cDNA from Peach

The ACC synthase is the key enzyme in ethylene biosynthesis and fruit ripening. To study the mechanism of ACC synthase in peach Prunus persica (L.) Batsch) fruit ripening, we cloned a full_length cDNA of ACC synthase pacs from peach using 5′/3′ RACE PCR. The nucleic acid sequence of pacs was 1 848 bp, containing 177 bp of 5′untranslated sequence, 1 449 bp of an open reading frame, and 219 bp of 3′untranslated sequence (excluding the stop codon TAA). The pacs open reading frame encoded a 483_amino acid polypeptide with a predicted size of 54 kD and a calculated PI of 6.43. The deduced protein from ACC synthase cDNA pacs had 65%, 70%, 75%, and 90% homology with the other deduced proteins from tomato (S19677), plum (AB031026), papaya (U68216) and apple (AB034993), which contained the active site of ACC synthase SLSKDMGFPGFR conserved among these plant ACC synthases. RNA_based PCR amplification combined with hybridization analysis with pacs and another ACC synthase cDNApacs12 (AF467782) cloned by us before as probes, indicated that expression patterns of both clones were very similar. mRNAs of both clones expressed in the alabastrum and petal, and were induced after ethylene treatment. Wounding and IAA treatments could induce ACC synthase expression of both clones in the leaves. However, the wounding treatment of leaves has induced more abundant pacs ACC synthase expression than that ofpacs12. Pacs mRNA expressed in both green mature and ripening fruit, whilepacs12mRNA was little or undetectable in green mature fruit, but apparent in ripening fruit. Both clone mRNAs accumulated more in leaves (following wounding and IAA treatments) and flowers than in fruits.

Genetic Transformation in Triticeae Crops

Wheat, triticale, tritordeum, barley, oat and rye are the most important crops in human consumptions and industry in the world. Transformation technology supplies a new source of improving Triticeae crops. In the past decade, transformation of wheat crops has considerably progressed. Many transgenic plants of Triticeae crops with various genes were produced via nricroprojectile bombardment, Agrobacterium-mediated transformation, PEG-uptake DNA technique, electroporation, microinjection, injection inflorescence and silicone carbide. Integration and expression of transgenes, inheritance and variation of transgenic plants have been studied. Technical improvements of genetic transformation for wheat crops will be extensively useful in commerce and benefit significantly to human being in the world.

The Promoter Hypermethylation of DAPK Gene and pl6 Gene in Sera from Chinese Non-small Cell Lung Cancer Patients

Objective： To evaluate the clinical significance of the aberrant methylation of DAPK gene and p16 gene in sera from 65 NSCLC patients from Nanjing General Hospital of Nanjing Command, China. Methods： A methylation-specific PCR （MSP） was performed for the detection of promoter hypermethylation of DAPK gene and p16 gene in blood DNA from 65 cases of NSCLC, and to analyze the relation of the aberrant methylation of DAPK gene and p16 gene and the clinicopathological data. Results： 30.8% （20/65） of the sera from 65 cases of NSCLC showed hypermethylation for DAPK promoter and 43.1% （28/65） the same for p16 promoter, whereas no methylated DAPK gene promoter and p16 gene promoter were found in sera from the patients with lung benign diseases and normal controls. Methylated DAPK gene promoter and p16 gene promoter in sera were not closely correlated with the pathological classification, stage, metastasis and differentiation in NSCLC. Conclusion： Detection of the aberrant methylation of DAPK gene and p16 gene in blood DNA from NSCLC patients might offer an effective means for the earlier auxiliary diagnosis of the malignancy.

Extracellular Calmodulin Induces rbcS-3A Gene Expression in Suspension Culture of Tomato Cells

利用番茄 (LycopersicumesculentumMill.)悬浮培养细胞为材料 ,以3 2 P标记的寡核苷酸 (40bp)为探针检测了细胞外钙调素对番茄细胞rbcS_3A及rbcS_3C基因表达的影响。当向暗中培养的番茄悬浮细胞 (第 7天 )中加入外源纯化钙调素 (10 -7mol/L)并处理 2 4h后 ,rbcS_3A基因的表达明显增加 ,而相同浓度的S_10 0蛋白和BSA则没有作用。加入外源钙调素 (10 -7mol/L)对rbcS_3C基因的表达没有影响。上述结果表明细胞外钙调素对暗中培养的番茄rbcS基因的表达有调控作用 ,并且这种调控作用具有亚型特异性 ,即对番茄rbcS_3A基因的表达有诱导作用 ,而对番茄rbcS_3C基因的表达没有作用。

Molecular Cloning and Expression of Carotenogenic Genes in Yellowish and Mutant Whitish Loquat (Eriobotrya japonica) Fruits

ObjectiveThe study aimed to explore the factors regulating carotenoid accumulation in flesh color. MethodA loquat mutation （red-or orange-fleshed plant emerged a bud mutation of white-flesh in trunk） was used as material; HPLC analysis of β-carotene content was conducted. ResultThe β-carotene concentration in the flesh of wild and mutant types was 60.9 and 4.6 μg/g fresh weight, respectively. According to the conserved regions of genes from rose family genome, carotenogenic gene fragments in wild and mutant types were obtained. No nucleotide variation of the carotenogenic gene fragments was observed between wild and mutant genome. Real-time quantitative polymerase chain reaction （Q-PCR） was compared and one carotenogenic gene, β-ring hydroxylase （HYB） were considerably suppressed in mature mutant loquat fruits compared with that in wild. The other six carotenogenic genes were also expressed but the expression patterns appeared to be not correlated with the amount of β-carotene concentration in wild loquat flesh. ConclusionThe mutant whitish loquat lacks the ability to synthesize β-carotene because of the transcriptional down-regulation of carotenogenic gene HYB.

Cloning and Prokaryotic Expression of NS1 Gene of Porcine Parvovirus (PPV) SD1 Strain

[Objective] The research aimed to provide the theoretical basis for establishing a rapid diagnosis method for porcine parvovirus（PPV）. [ Method] One pair of primers were designed according to PPV genome sequences on GenBank website and the sequences of prokaryotic expression vector pET30a （ ＋ ） with multiple cloning sites. The whole sequence of NS1 gene in PPV SD1 strain was amplified by using PCR technology and the positive recombinant plasmid was analyzed by sequencing and homology comparison. The prokaryotic expression recombinant plasmid PET30a/NS1 was constructed to make its induction expression in Escherichia coll. [ Result] The target fragment with the length of 2 208 bp was obtained from PCR amplification. The nucleotide homologies between the cloned NS1 gene and the reported relevant PPV genes were from 97.3 % to 99.4 %, which indicated that NS1 gene had high conservation. But it had a 12-basepair successive deletion near the hydroxyl end. The cloned PPV NS1 gene was successfully expressed in prokaryotic cell, and its expression products existed mostly in inclusion bodies. [ Conclusion] The results of SDS-PAGE detection showed that the molecular weight of PPV NS1 protein was 86 KD.

The Inheritance and Expression of cry1A Gene in Transgenic Maize

We investigated the inheritance and expression of cry1A gene in transgenic maize ( Zea mays L.) by Southern blotting analysis and enzyme_linked immunosorbent assays (ELISA). The results showed that cry1A had been transmitted to progeny of transgenic maize as a single gene. Contents of cry1A insecticidal protein were significantly different among transgenic maize lines and various tissues of the same transgenic lines. High expression of cry1A protein occurred in green tissues, such as leaf and husk leaf, and low expression occurred in pith, tassel, ear pith, pollen and silk. The results also showed that the contents of cry1A insecticidal protein in leaves of transgenic maize increased with the advance of development and there was no significant difference in cry1A expression level among various generations of transgenic maize.

Application of GFP Gene in the Study of Insect-Resistant Transgenic Plants

用合成的cry1Ac基因与绿色荧光蛋白基因 (GFP)构成融合蛋白基因 ,然后和改造的GNA基因构建双价抗虫基因植物表达载体pBGbfg ,经根癌农杆菌介导转化了烟草。在紫外灯照射下 ,观察到转基因植株叶片中有较强的绿色荧光 ;经抗虫试验、PCR、Southernblot和Westernblot等检测 ,表明该重组植物表达载体能够在转基因植物中有效表达外源基因 ,转基因植株绿色荧光的表型与其抗虫性密切相关。从而成功地建立了以绿色荧光蛋白基因与抗虫基因组成的融合基因转化系统 ,简化了抗虫转基因植物筛选程序 ,有助于快速获得双价抗虫转基因植株。

Transcriptional Regulation of 10 Mitochondrial Genes in Different Tissues of NCa CMS System in Brassica napus L. and Their Relationship with Sterility

Northern blot analysis was conducted with mitochondrial RNA from seedling leaves, floral buds, and developing seeds of NCa CMS, maintainer line and fertile F1 using ten mitochondrial genes as probes. The results revealed that 9 out of the 10 mitochondrial genes, except for atp6, showed no difference in different tissues of the corresponding materials of NCα CMS system and that they might be constitutively expressed genes. Eight genes, such as orf139, orf222, atpl, cox1, cox2, cob, rm5S, and rm26S, showed no difference among the three tissues of all the materials detected. So the expression of these eight genes was not regulated by nuclear genes and was not tissue-specific. The transcripts of atp9 were identical among different tissues, but diverse among different materials, indicating that transcription of atp9 was neither controlled by nuclear gene nor tissue-specific. Gene atp6 displayed similar transcripts with the same size among different tissues of all the materials but differed in abundance among tissues of corresponding materials and its expression might be tissue-specific under regulation of nuclear gene. Moreover, three transcripts of orf222 were detected in the floral buds of NCa cms and fertile F1, but no transcript was detected in floral buds of the maintainer line.The transcription of orf139 was similar to that of orf222 but only two transcripts of 0.8 kb and 0.6 kb were produced. The atp9 probe detected a single transcript of 0.6 kb in NCa cms and in maintainer line and an additional transcript of 1.2 kb in fertile F1. The relationship of expression of orf222, orf139, and atp9 with NCa sterility was discussed.

Relationship between the Expression of MTA1 Gene and Invasion and Metastasis of Human Osteosarcoma Cells

Objective： To compare the expression level of metastasis associated-1 （MTA1） in the higher and lower metastasis sublines of human osteosarcoma cells （MG63）, and to investigate the relationship between the expression level of MTA1-EGFP and in vitro invasion and metastasis of human osteosarcoma cells. Methods： The expression level of MTA1 in two sublines of MG63 cells was detected by semi-quantitative RT-PCR, and cell invasion assay and cell proliferation assay were used to evaluate the invasive capacity in vitro in two sublines. The lower metastasis line of MG-63 cells were transfected with MTA1-EGFP full-length cDNA expression plasmid by lipofectamine. The changes of the MTA1-EGFP expression and in vitro invasion potential were measured after transfection. Results： M8 subline expressed significantly higher level of MTA1 than that of M6 subline by RT-PCR. The invasive potentials of low metastasis MG63 cell line were increased after MTA1 gene transfection. Conclusion： There may be a relationship between MTA1 and invasive potentials of human osteosarcoma cells, and MTA1 may play a role in the molecular mechanism of tumor metastases and be a potential target for gene therapy of osteosarcoma. Further studies of MTA1 in human ostersarcoma cell metastasis are needed.

Expression of a Wheat S-like RNase (WRN1) cDNA During Natural-and Dark-induced Senescence

An S-like RNase cDNA had been isolated from common wheat (Triticum aestivum L). The transcription of WRN1 mRNA was down-regulated by natural- and dark-induced senescence. But it was not senile-tissue-specific. As the two key histidine residues were replaced, WRN1 may not be active as RNase. Southern blotting analysis showed that WRN1 exists as one of a small gene family in common wheat genome.

Expression of the Glypican-3 Gene in α-fetoprotein-negative Human Hepatocellular Carcinoma

Objective： To investigate the expression of Glypican-3 gene in （α-fetoprotein （AFP）-negative hepatocellular carcinoma （HCC） and clarify whether Glypican-3 expression is a useful parameter for the diagnosis of hepatocelhllar carcinoma （HCC）, especially for AFP-negative ones. Methods： Forty-one specimens of AFP-negative hepatocellular carcinoma and para-carcimoma tissue were studied for the expression of Glypican-3 by Northern blot. The expression of Glypican-3 protein was detected immunohistochemically with specific polyclonal antibody. Results： Northern blot analysis indicated that the expression of Glypican-3 mRNA was intensively detected in 30 of 41 AFP-negative HCC （73.17%）. In contrast, Glypican-3 mRNA was only weakly detected in 4 of the surrounding non-tumor liver tissues. There was significant difference in the Glypican-3 mRNA expression in large tumors （〉5 cm） （79.31%） and in small tumors （〈5 cm） （41.67%） （P〈0.01）. Gypican-3 mRNA was more frequently overexpressed in poorly differentiated HCC than in well differentiated ones （76.47% vs. 42.86%, P〈0.05）. The Glypican-3 expression was not correlated with age. sex. ttbsAg seropositivity, fibroeapsule, portal venous embolus and intrahepatic metastasis. The overexpression of Glypican-3 protein in HCC was targeted in tumor cells, not in bile duct cells and other interstitial cells. Conclusion： Glypican-3 was specially overexpressed in AFP-negative HCC, and its expression was closely correlated with the tumor size and tumor grade. Glypican-3 gene may play important roles in hepatocareinogenesis, and can be used as a new biochemical marker of HCC, especially for AFP-negative HCC.

The Relationship between Methylation and Expression Defect of Tumor Suppressor Gene p16INK4A in Epithelial Ovarian Cancer

Objective： To evaluate the expression of p16INK4A gene in ovarian cancer and analyze the relation between this alteration and the promoter methylation of p16INK4A DNA. Methods： Seven ovarian cancer cell lines and eighteen ovarian cancer specimens were selected for the study. Genomic DNA and RNA were extracted from fresh tissues and cell lines, DNA was treated with sodium bisulfite and then analyzed with methylation-specific PCR （MSP） to detect p16INK4A methylation. The expression of p16INK4A mRNA was detected by reverse transcription-polymerase chain reaction （RT-PCR）. In addition, the proliferation of methylated cell lines before and after treatment of demethylating agent 5-Aza-2＇-deoxycytidine （5-ADC） was examined with 3-（4, 5-dimethylthiazol-2-yl）-2, 5-diphenyltetrazolium bromide （MTT） assay in vivo. Results： Compared with the control, the expression of p16INK4A mRNA decreased significantly or absolutely defaulted in 10 of 18 （55.56%） ovarian cancer specimens and 71.4% （5/7） ovarian cancer cell lines （P〈0.05）, and the expression of p16INK4A protein also decreased （P〈0.05）. The decrease of p16INK4A was due, in part, to p16INK4A methylation, which was found in the first exon of three cell lines and six ovarian cancer specimens and the rate was 42.86% and 33.33% in ovarian cancer cell lines and specimens respectively. All the methylated cells and tissues showed expression defect of p16INK4A, but the treatment of 5-ADC reactivated the expression of p16INK4A in methylated cells and decreased the proliferation of tumor cells in vitro and in vivo. Conclusion： The expression defect of p16INK4A gene possibly has an important role in the development of ovarian cancer, and this alteration is due, in part, to the methylation of the first exon in p16INK4A.