PCR检测HPV－18DNA

采用聚合酶链式反应（ＰＣＲ）技术,对ＨＰＶ－１８序列中引物ＨＰ＿１、ＨＰ＿２之间的片段（Ｆ）进行扩增,通过两组阴、阳性对照实验证明扩增片段的特异性。用不同Ｍｇ浓度的缓冲系统进行ＰＣＲ反应发现,缓冲系统中Ｍｇ浓度高低是影响ＨＰＶ－１８／ＨＰ＿１、ＨＰ＿２特异扩增的重要因素,高浓度Ｍｇ导致扩增特异性降低。对１７例宫颈癌组织ＤＮＡ进行ＰＣＲ检测,有９例检出Ｆ片段,其检出率是５３％,为ＨＰＶ－１８与宫颈癌的相关性提供证据。     

用DNA重组法表达丙肝病毒部分NS_4-NS_5基因

用ＤＮＡ重组法表达丙肝病毒部分ＮＳ_４－ＮＳ_５基因祁自柏,谷金莲,李河民（中国药品生物制品检定所,北京１０００５０）丙型肝炎病毒（ＨＣＶ）是一种ＲＮＡ病毒,为研究其复制机理,制备检测丙肝抗体的诊断试剂,需大量纯化的各种丙肝病毒蛋白,我们在国内首次用Ｄ...     

Enhancing PCR amplification and sequencing using DNA-binding proteins

The polymerase chain reaction (PCR) is a powerful core molecular biology technique, which when coupled to chain termination sequencing allows gene and DNA sequence information to be derived rapidly. A number of modifications to the basic PCR format have been developed in an attempt to increase amplification efficiency and the specificity of the reaction. We have applied the use of DNA-binding protein, gene 32 protein from bacteriophage T4 (T4gp32) to increase amplification efficiency with a number of diverse templates. In addition, we have found that using single-stranded DNA-binding protein (SSB) or recA protein in DNA sequencing reactions dramatically increases the resolution of sequencing runs. The use of DNA-binding proteins in amplification and sequencing may prove to be generally applicable in improving the yield and quality of a number of templates from various sources.     

A family of serine protease genes expressed in adult buffalo fly (Haematobia irritans exigua)

Gene fragments encoding serine proteases expressed in adult buffalo fly (Haematobia irritans exigua) were amplified from cDNA using generic oligonucleotide PCR primers, based on conserved residues surrounding the active-site His and Ser amino acids found in all serine proteases. The PCR product consisted of a broad band extending from about 450 by to 520 bp, which suggested that the PCR product actually consisted of numerous DNA fragments of slightly variable sizes. Seventeen independent clones of these fragments, each with an insert of approximately 480 bp, were digested with HaeIII. Comparison of restriction fragment patterns indicated that 13 of these clones harboured different PCR products. This was confirmed by DNA sequence analysis of 9 clones. Each of the sequenced clones contained an open reading frame which included structurally conserved regions characteristic of the serine protease superfamily. This study reveals the expression of a large and highly variable repertoire of serine proteases in adult buffalo fly. Importantly, these data also demonstrate the utility of such an approach in obtaining DNA probes for use in further investigations of gene family organization and expression, as well as providing recombinant antigens in the form of fusion proteins which may be used as candidates for vaccine production.     

Overexpression of cloned genes using recombinant Escherichia coli regulated by a T7 promoter: II. Two-stage continuous cultures and model simulations

A two-stage culture strategy was studied for continuous high-level production of a foreign protein in the chemically inducible T7 expression system. The first stage is dedicated to the maintenance of plasmid-bearing cells and the second stage to the target protein synthesis by induction of cells coming from the first stage. On entering the second stage, recombinant cells undergo a gradual induction of the target gene expression. These plasmid-bearing cells experience dynamic changes in intracellular compositions and specific growth rates with their individual residence times. Therefore, the overall cultural characteristics in the production stage are really averages of the contributions from the various cells with different residence times. The behavior of the two-stage culture is described by a model, which accounts for dynamic variations of cell growth and protein synthesis rates with cell residence times. Model simulations were compared with experimental results at a variety of operating conditions such as inducer concentration and dilution rate. This model is useful for understanding the behavior of two-stage continuous cultures. (c) 1993 John Wiley & Sons, Inc.     

Kinetics of cell growth and heterologous glucoamylase production in recombinant Aspergillus nidulans

In the work, a study of cell growth and the regulation of heterologous glucoamylase synthesis under the control of the positively regulated alcA promoter in a recombinant Aspergillus nidulans is presented. We found that similar growth rates were obtained for both the host and recombinant cells when either glucose or fructose was employed as sole carbon and energy source. Use of the potent inducer cyclopentanone in concentrations greater than 3 mM resulted n maximum glucoamylase concentration and maximum overall specific glucoamylase concentration over 80 h of batch cultivation. However, cyclopentanone concentrations in excess of 3 mM also showed an inhibitory effect on spore germination as well as fungal growth. In contrast, another inducer, threonine, had no negative effect on spore germination even when concentrations of up to 100 mM were used with either glucose or fructose as carbon source. Glucoamylase synthesis in the presence of glucose plus either inducer did not begin until glucose was totally depleted, suggesting strong catabolite repression. Similar results were obtained when fructose was employed, although low levels of glucoamylase were detected before fructose depletion, suggesting partial catabolite repression. The highest enzyme concentration (570 mg/L) and overall specific enzyme concentration (81 mg/g cell) were observed in batch culture when cyclopentanone was the inducer and fructose the primary carbon source. A maximum glucoamylase concentration of 1.1 g/L and an overall specific glucoamylase concentration of 167 mg/g cell were obtained in a bioreactor using cyclopentanone as the inducer and limited-fructose feeding strategy, which nearly doubles the glucoamylase productivity from batch cultures. (c) 1993 John Wiley & Sons, Inc.     

Comparison of three different promoter systems for secretory alpha-amylase production in fed-batch cultures of recombinant Saccharomyces cerevisiae

Cloned gene expression in recombinant Saccharomyces cerevisiae 20B-12 containing three different plasmids was compared in batch and fed-batch cultures. The plasmids pNA3, pNA7, and pNA9 contain the alpha-amylase gene under the control of SUC2, PGK, and GAL7 Promoters, respectively. The synthesis of alpha-amylase was therefore induced by low glucose concentration for the SUC2 and PGK promoters, and by galactose for GAL7 promoter. The specific cell growth rates were similar among cells harboring the three different plasmids; they decreased from 0.35 to 0.38 h(-1) during the cell growth phase to 0.03 to 0.06h(-1) during the production phase. The secretory alpha-amylase activity of cells harboring plasmid pNA7 was 129 U/mL in fed-batch culture, which was 1.4 and 2 times as high as the activities of cells harboring plasmids pNA3 and pNA9, respectively. The secretion ratios (amount of extracellular alpha-amylase activity/amounts of total alpha-amylase activity) of cells harboring plasmids pNA3, pNA7, and pNA9 were 91.4%, 94.5%, and 95.3%, respectively. (c) 1993 Wiley & Sons, Inc.     

Metabolic engineering of Clostridium acetobutylicum ATCC 824 for increased solvent production by enhancement of acetone formation enzyme activities using a synthetic acetone operon

The ability to genetically alter the product-formation capabilities of Clostridium acetobutylicum is necessary for continued progress toward industrial production of the solvents butanol and acetone by fermentation. Batch fermentations at pH 4.5, 5.5, or 6.5 were conducted using C. acetobutylicum ATCC 824 (pFNK6). Plasmid pFNK6 contains a synthetic operon (the "ace operon") in which the three homologous acetone-formation genas (adc, ctfA, and ctfB) are transcribed from the adc promoter. The corresponding enzymes (acetoacetate decarboxylase and CoA-transferase) were best expressed in pH 4.5 fermentations. However, the highest levels of solvents were attained at pH 5.5. Relative to the plasmid-free control strain at pH 5.5, ATCC 824 (pFNK6) produced 95%, 37%, and 90% higher final concentrations of acetone, butanol, and ethanol, respectively; a 50% higher yield (g/g) of solvents on glucose; and a 22-fold lower mass of residual carboxylic acids. At all pH values, the acetone-formation enzymes were expressed earlier with ATCC 824 (pFNK6) than in control fermentations, leading to earlier induction of acetone formation. Furthermore, strain ATCC 824 (pFNK6) produced butanol significantly earlier in the fermentation and produced significant levels of solvents at pH 6.5. Only trace levels of solvents were produced by strain ATCC 824 at pH 6.5. Compared with ATCC 824, a plasmid-control strain containing a vector without the ace operon also produced higher levels of solvents [although lower than those of strain ATCC 824 (pFNK6)] and lower levels of acids. Strains containing plasmid-borne derivatives of the ace operon, in which either the acetoacetate decarboxylase or CoA-transferase alone were expressed at elevated levels, produced acids and solvents at levels similar to those of the plasmid-control strain. (c) 1993 John Wiley & Sons, Inc.     

A two-stage bioreactor system for the production of recombinant proteins using a genetically engineered baculovirus/insect cell system

A two-stage bioreactor scheme was developed for the large-scale production of recombinant proteins using a genetically engineered baculovirus/insect cell system. The first bioreactor was employed for cell growth and the second for cell infection. Silkworm Bm5 cells were infected with a recombinant baculovirus, BmNPV/P5.cat, containing a bacterial chloramphenicol acetyltransferase (CAT) gene under the control of the polyhedrin gene promoter of Bombyx mori nuclear polyhedrosis virus (BmNPV). This recombinant baculovirus has been used as an expression vector for the production of recombinant CAT enzyme. A specific productivity of 82 to 90 mug CAT/(10(6) cells) was obtained using the BmNPV/Bm5 expression system, a yield similar to that achieved using the AcNPV/Sf expression system. Repeated infection of high-density cell cultures did not reduce the specific productivity of the CAT enzyme. Most importantly, the problems associated with the infection of high-density cell cultures were resolved by means of controlled infection conditions and appropriate replenishment of spent culture medium following infection. The glucose uptake rate by the cells following infection was 50% higher than that by the cells before infection. Not only did the infection of high-density cell cultures result in consistent yields of 250 mg/L of CAT enzyme, but also the two-stage bioreactor system was proven to be reliable for a long-term operation beyond 600 h. (c) 1993 John Wiley & Sons, Inc.