Abstract:A novel coronavirus has been identified to be associated with the newly outbreak of severe acute respiratory syndrome (SARS). To date 12 whole genome sequences from various samples are available in public-domain database. Spike protein of coronavirus is considered to be the major antigen and contains many potential antigenicity domains. The relatively low mutation rate in spike protein provides high opportunity for effective vaccine development. Since inactivated or attenuated coronavirus holds some potential limitations and risks to prepare and to inoculate, the current best hope for protection is to combine a protein vaccine (i.e., a purified SARS coronavirus spike protein) with a “vectored” vaccine, consisting of a plasmid or a harmless virus, such as recombinant adenovirus which has been genetically engineered to produce coronavirus spike protein.

Abstract:RNA interference (RNAi) is the process of sequence-specific, post-transcriptional gene silencing in animals and plants, initiated by double-stranded RNA (dsRNA) that is homologous in sequence to the silenced gene. In mammal cells, small interfering RNA (siRNA) duplexes can induce RNAi potently, which may provide a new approach to the therapeutics of certain diseases. Focusing on the five genes which coding five crucial proteins of SARS coronavirus(SARS-CoV) respectively, 348 siRNA candidate targets were obtained following bioinformatic methods. In theory, potent siRNA duplexes specifically suppress expression of their corresponding SARS-CoV target gene, while have no influence on the normal expression of human gene. It would lay a foundation for the further experimental researches on the siRNA-like drug design for the SARS-CoV.

Abstract:Acid-sensing ion channels (ASICs) are ligand-gated ion channels activated by extracellular proton. To date, six members of ASICs family have been identified. They are widely expressed in the peripheral and central nervous system. Using gene knockout techniques, ASICs are demonstrated to be involved in sensory perception as touch, pain, sour taste, learning and memory as well as some pathological conditions. ASICs can be modulated by neuropeptide, temperature, some metal ions and ischemia-related compounds, integrating multiple extracellular signals to function their roles.

Abstract:The rapidly accumulating data of sequenced microbial genomes allows to conduct systematic studies on microbial gene regulation as well as function. As proteins are often the functional molecules, proteomics is particularly booming in the functional studied of microbial genomics. Using comparative studies as the powerful tools, a fundamental principle in microbial proteomics is to elucidate and to understand the gene expression levels in different microorganisms or under different growth conditions. It is very obvious that the quantitative analytical techniques are highly required for comparative proteomics. The current status of the approaches applied to quantitative analysis of microbial proteomics were reviewed.

Abstract:Protein phosphorylation is a universal regulatory way in organism, and it is a key event in cell signal transduction. Mass spectrometry has emerged as an increasingly viable tool for this task. The methodologies currently available for the analysis of phosphoproteins by mass spectrometry were summarized, including enrichment of compounds of interest using immobilized metal affinity chromatography, antibody and chemical tagging techniques, detection of phosphopeptides using mass mapping and precursor ion sequencing, localization of phosphorylation sites by peptide sequencing, and quantitation of phosphorylation by the introduction of mass tags. Despite the variety of powerful analytical methods that are now available, complete characterization of the phosphorylation state of a protein isolated in small quantities from a biological sample remains far from routine.

Abstract:Neurodegenerative disease is a kind of neural system disease which is characterized by degenerative pathological changes of neuron, the consequent behavioral abnormal and even individual death at last. Drosophila, with uniquely powerful molecular genetics, has been a competent model to research human neurodegenerative disease. Screening the genes of Drosophila could not only define the molecular pathway that underlies the neurodegenerative process, but also provide valuable drug targets by consequently studying the control genes and gene products. All of these will uncover the novel means of preventing and curing human neurodegenerative disease.

Abstract:As the first RNA catalyst (ribozyme) discovered in nature twenty years ago, group Ⅰ intron has been extensively studied to understand catalysis, structure and folding of the catalytic RNA, which fundamentally contributes to the current understanding of RNA structure and function. Most of the major topics in group Ⅰ intron study, emphasizing on the tertiary structure and folding issues that has bloomed in the past several years are reviewed.

Abstract:Recently, a class of ～21 nucleotides(nt) small RNA have been discovered in many eukaryotes, termed miRNAs (microRNAs), which were first identified as key temporal regulators in development. So far, large quantities of studies have revealed that miRNAs have played important roles in genetic control at many different levels and rearrangement of genome. Besides, its association with siRNA (small interfering RNA) previously discovered in RNAi (RNA interference) in the further researches becomes much closer than it has ever been considered. Its pathway directing translational repression, the surprisingly high conservation of certain miRNA, the mechanism of process of mature miRNA and genetic regulation compared with that of siRNA were focused. Finally several discussions arising people's interests caused by the discovery of miRNA are made.

Abstract:Molecular cloning has identified three distinct caveolin genes: caveolin-1, caveolin-2, and caveolin-3. Caveolin-1 and caveolin-2 have been mapped to a common locus in chromosome 7q31.1, that is a possible candidate for a tumor suppressor gene postulated in this region. Caveolin-1 assumes an unusual topology. A central hydrophobic domain is thought to form a hairpin-like structure within the membrane. As a consequence, both the N-terminal domain and the C-terminal domain face the cytoplasm, thereby forming a caveolin-rich scaffold. Caveolin-1 and caveolin-2 proteins interact with themselves to form homo- and hetero-oligomers and are thought to be the driving force for caveolae formation. They are most abundantly expressed in adipocytes, endothelial cells and fibroblastic cell types, whereas caveolin-3 is muscle-specific. Both in vitro and in vivo experiments show the transformation suppressor activity of caveolin-1, indicating that caveolin-1 may provide a necessary brake in signal transduction. The targeted disruption of caveolin-1 in mice results in impaired nitric oxide and calcium signaling in the cardiovascular system, and displays thickening of alveolar septa caused by uncontrolled endothelial cell proliferation. Caveolin-1-deficient mice are lean, resistant to diet-induced obesity, and show hypertriglyceridemia with adipocyte abnormalities. Caveolin-2-null phenotypes are identical to the ones that have been reported for caveolin-1-null mice in lung function. Caveolin-3 is a component of the dystrophin complex, and might be relevant to Duchennes and other muscular dystrophies. The loss of caveolin-3 expression in mice is sufficient to induce a molecular program leading to cardiac myocyte hypertrophy and cardiomyopathy.

Abstract:A novel model of TRAIL-induced apoptosis of Novikoff cells was constructed based on the observation using molecular fluorescence labelling and confocal microscopic techniques. The mechanism of TRAIL-induced apoptosis and the role of Ap5A ［P¹, P⁵-Di（adenosine-5′）pentaphosphate］ during this process was investigatived. The results revealed that: 1) TRAIL-induced apoptosis is both dosage- and time- dependent, which correlates with the remarkable increase of ［Ca²⁺］ in Novikoff cells; 2) Ap5A retards the TRAIL-induced apoptosis as well as down-regulates ［Ca²⁺］ in Novikoff cells. These observations indicate that the mechanism of TRAIL-induced apoptosis and Ap5A retards it through down and up-regulating intracellular calcium concentration respectively.

Abstract:Chicken counterpart of interleukin-2 (chIL-2) is discovered recently which shows great potential in immunotherapy and immunoadjuvant applications. In view of its immunomodulating effects and involvement in disease resistant traits, this study compared the sequence relatedness and the genetic evolution of chIL-2 genes of local breeds with other geographical isolates. The chIL-2 genes from two Chinese local breeds (Xianju and Silky) and a commercial broiler breed (Avian) were amplified and cloned from Con A-stimulated chicken spleen lymphocytes. Sequence comparison revealed a similar genetic organization among the breeds with a coding region of 429 bases, a 5′-terminal untranslated region (UTR) with 17 nucleotides in length and an UTR with 285 nucleotides with five repetitive sequence of ATTTA at 3′-terminal. The deduced amino acid sequences were compared with strains Kestrel, Obese and SC Leghorn chickens from GenBank. Results showed that Xianju, Silky and Kestrel breeds shared an identical chIL-2 gene while the chIL-2 from Avian commercial broiler, Obese and SC Leghorn chickens was identical. In addition, a hypermutation region between residue 28 to 32 was identified. Phylogenetic analysis grouped Xianju, Silky and Kestrel, and Avian, Obese and SC into a separate group. Furthermore, a mutation from glutamic acid (E) to glycine (G) was found at the conserved residue 133 in Chinese official chicken breed-Silky chicken.

Abstract:Brief periods of ischemia can protect the heart from a subsequent longer coronary artery occlusion, the phenomenon called ischemic preconditioning. The mechanism of ischemic preconditioning are not well understood. A number of genes have been shown to play roles in cytoprotective effect of ishemic preconditioning, but there are clearly many missing elements to be found. Suppression subtractive hybridization was used to construct cDNA libraries enriched for genes up-regulated during ischemic preconditiong. After being confirmed by reverse Northern dot blot for differential expression, the selected genes were sequenced and searched in GenBank for homology analysis. Many nuclear-encoded genes that were up-regulated during ischemic preconditioning participate in cytoprotection. The 18 novel ESTs were banked into GenBank with accession numbers. The specificity of this response was confirmed by RT-PCR and Northern blot. Understanding the genes up-regulated during ischemic preconditioning may open new avenues for therapy in ischemic heart disease.

Abstract:The relationship between sulfated lactosyl ceramide expressional level and hepatocellular carcinoma metastasis potential was investigated. By means of immunofluorescent staining and cell ELISA with monoclonal antibodies against sulfated lactosyl ceramide or galactocerebroside, it was observed that the expression of sulfated lactosyl ceramide in high metastasis hepatocarcinoma cells was significantly higher than those in low metastasis hepatocarcinoma cells. After the hepatocarcinoma cells with high metastatic potential were treated with 10 μmol/L retinoic acid, the level of sulfated lactosyl ceramide on the cell surface was significantly reduced (P＜0.05) to the level closing to the low metastatic hepatocarcinoma cells. The level of galactocerebroside which was the precursor of sulfated lactosyl ceramide did not change significantly. This indicated that the sulfation was inhibited after retinoic acid treatment. Therefore the cerebroside sulfotransferase gene were transferred into hepatocarcinoma cells and selected sulfated lactosyl ceramide-highly-expressed clones. Through adhesive assay, it was observed that the control cells were quite poor in adhesive to vitronectin and laminin, the transfected cells were significantly enhanced in the adhesion to vitronectin and laminin. After 45 minute incubation, the adhesive percentage of the cells was 3～4 time higher in CST-transfected cells than those in control cells. By animal experiments there were more metastatic foci in the liver or lymph nodes in CST-transfected groups than the control (P＜0.05). The results suggested that the high expression of SM3 in hepatocarcinoma cells promoted metastasis in nude mice and enhanced the adhesion to vitronectin.

Abstract:Nasopharyngeal carcinoma (NPC) is a rare malignancy tumor in most parts of the world, while with high incidence rate in the south of China. NOR1, a NPC down-regulated gene, was newly-cloned using cDNA micro-array. NOR1 gene has an oxidored-nitro domain predicted by bioinformatics. The latest results suggest that the NOR1 gene may participate in the metabolism of chemical carcinogen such as nitrosamine in vivo. Genotype of coding region single nucleotide polymorphisms (cSNPs) in NOR1 gene were performed by sequencing in 144 unrelated NPC subjects and 144 control subjects which matched in age, sex and residence. Association analysis suggests that there is linkage disequilibrium between the 2 cSNPs, both of them associated with NPC. And the relative risk of 2 cSNPs and their haplotypes were 1.36, 1.64 and 1.37 respectively. Both of the two SNPs could change the sequence of NOR1 protein, which might influence it's structure and function. The results indicated that the SNPs in NOR1 gene may play a certain role in carcinogenesis and development of NPC.

Abstract:Eight weeks old db/db mice were treated with or without rhein at a dosage of 150 mg/kg for 12 weeks. Kidney's total RNA was extracted and renal gene expression profiles were tested using the gene chip GM-U74A from Affymetrix company. Among the total 12 437 tested genes, 1 085 genes were down-regulated and 37 genes were up-regulated in untreated db/db mice when compared with normal control. Among them, 166 genes were down-regulated and 29 genes up-regulated more than 2 fold. It was found that 384 genes were down-regulated and 155 genes up-regulated in rhein treated db/db mice as compared with untreated db/db mice. About 47 genes were down-regulated and 30 genes up-regulated more than 2 fold. Then, an EST, which was down-regulated by about 2 fold in db/db mice and recover to normal level in rhein treated mice, was further analyzed by bioinformatical method and proved to be part of “REKEN cDNA 0610006H10” gene, which function is unknown. After its expression level was further proved by RT-PCR, diabetic nephropathy relative gene,“REKEN cDNA 0610006H10” gene, was cloned for further study.

Abstract:DNA chip and DNA computing are new research areas in biology science and information science separately. The essential characteristic of both is the massive parallel of obtaining and managing information. The 0-1 programming problem is an important problem in opsearch and has very widespread application. But up to now, there does not exist any good algorithm yet. A new DNA computing model is provided to solve a 0-1 planning problem based on DNA chip. The method has some significant advantages and the result suggests the potential of DNA chip used as a DNA computer chip.

Abstract:The stochastic master equation approach to molecular motor's directed motion is used and a periodic one-dimensional four-state stochastically unequal interval hopping model is studied. The drift velocity V, the diffusion constant D and the randomness parameter r of the steady state are all be obtained. By comparing the simulated curve with experiment about drift velocity V, diffusion constant D and randomness parameter r versus ［ATP］ and external load F, the kinetic behaviors of a molecular motor under external load F are qualitatively analyzed.

Abstract:To evaluate its safety, biological activity and immunogenicity of the recombinant H.pylori (Hp) adhesin conservation region(AB) in vitro so that to determine the feasibility of AB in Hp vaccination. ELISA assay was used to measure AB-specific antibody in serum of Hp infected patients. The proliferation of T cell in response to AB was examined by MTT test. Flow cytometry was used to evaluate the increase in FasL expression on T cells under the stimulation of Hp AB,T cell apoptosis induced by AB was detected by DAP assay.The effect of anti-AB serum on Hp binding of human gastric carcinoma cell lines was determined by light microscopy. Antibodies were detected in sera samples from 55 patients by ELISA, with RUT as parallel compare. Kappa value is 0.76. The low dose AB was capable of stimulating proliferation of T cells from Hp positive subjects. The effect of AB was significantly lower in both the induction of apoptosis and FasL expression of T cells than that of ATCC26695. Anti-AB serum could partially inhibit Hp binding to gastric epithelial cells. Under light microscopy, the adhesion of Hp to MGC-803 was significantly inhibited when pretreated the bacteria with anti-AB rabbit serum, compared with negative control which pretreated with pre-immunized rabbit serum. AB was a safe, immunogenicity thallus component, which can stimulate humoral and cellular immunity.Its antibody was capable of preventing the binding of Hp to gastric epithelial cell.

Abstract:hhlim is a new heart-related gene cloned from human embryonic heart whose product participates in transcriptional regulation and cell development as a kind of transcriptional factor. Over expression of hhlim gene using a recombinant plasmid was sufficient to induce a greater than 2.49 fold increase in cardiac myocyte area compared with that untransfected with hhlim. RT-PCR and Western blot testified that transfection of hhlim into the cardiac myocyte could induce skeletal α-actin over expression and trigger the expression of embryonic related gene-BNP, which is related to the cardiac hypertrophy. Antisense hhlim expression plasmid was constructed for analyze of hypertrophy in cultured neonatal cardiomyocytes. Endothelin-1 can induce cardiomyocyte hypertrophy. Cardiac myocyte treated by ET-1 was transfected with antisense hhlim palsmid. Western blot and RT-PCR analysis demonstraned that antisense hhlim restrained the increased cell surface area induced by ET-1, or increases expression of α-actin and BNP. Individual expression vectors for hhlim, Nkx2.5 and GATA-4 could enhance BNP reporter gene expression in cardiac myocytes. Cotransfection of hhlim and Nkx2.5 produced additive luciferase expression. The results demonstrate that hhlim protein is capable of initiating the hypertrophic response in cultured cardiac myocyte by activing BNP gene expression directly and indirectly.

Abstract:The kinetics of asymmetric reduction of acetyltrimethylsilane and its carbon analogue catatlyzed by horse liver alcohol dehydrogenase were explored. It has been found that the relation between substrate concentration and initial reaction rate was in accordance with the Michaelis-Menten equation when enzyme concentration was below 150 mg/L and the K_m, v_max and E_a of HLADH-catalyzed asymmetric reduction of acetyltrimethylsilane were 2.67 mmol/L, 0.118 mmol/(L·min·mg) and 37 kJ/mol respectively, while the corresponding parameters for its carbon analogue were 3.56 mmol/L, 0.084 mmol/(L·min·mg) and 61 kJ/mol.

Abstract:E-cadherin-negative human breast carcinoma cell lines, MDA-MB-231 and MDA-MB-435 were transfected with wild-type E-cadherin cDNA. Flow cytometry showed that E-cadherin-positive transfectants grew slower than the control cells and more cells were relayed in G0/G1 phase. Western blot showed that it was due to down-regulation of protein concentration of cyclin D1 and β-catenin, the cyclin D1 gene transcriptional regulator. At the meantime, PKB protein level, which can inhibit β-catenin destruction through GSK-3β, was also down-regulated. As the PKB activators, FAK and ILK protein levels were decreased and PKB inhibitor, PTEN was increased by positive expression of E-cadherin. Therefore, E-cadherin can inhibit PKB activity by down-regulation of FAK, ILK and up-regulation of PTEN. As a result, β-catenin and cyclin D1 protein level increased and more cells were retarded in G0/G1 phase.

Abstract:A new galactose-specific lectin (MPL) from Musca domestic pupae was isolated. The purification procedure entailed extraction with aqueous buffer, GRBC (glutaraldehydeized red blood cells) adsorption, affinity chromatography on Sepharose-4B and gel-filtration on SephadexG-200. By SDS-PAGE, purified MPL yielded three bands, with a total molecular mass of 84 ku. By gel-filtration, the molecular mass of MPL was determined to be 86 ku. MPL exhibits high affinity towards D-galactose. The hemagglutination activity of MPL was independent of Ca²⁺, heat liable and stable in the range of pH 6～9. Antibacterial activity of MPL has been observed against Escherichia coli, Bacillus subtilis and Salmonella typhi.

Abstract:Vasostatin gene was amplified from a human liver cDNA library by PCR method. The fragment was cloned into the pUC19 vector and sequenced. By inserting the vasostatin fragment into the pQE-30 vector, the recombinant pQE-30/vaso plasmid was constructed. After it was transformed into E.coliM15, the recombinant proteins were expressed successfully when induced with IPTG. The expressed recombinant protein accounted for more than 50% of total bacterial proteins. The expressed products formed inclusion body in E.coli. After extracted from bacterial cells and washed, it was dissolved in solution containing 8 mol/L urea and then purified by using immobilized metal ion affinity chromatography (IMAC) effectively with a purity of over 95%. Then the recombinant protein was renatured after the denaturants were removed gradually by dialysis. The protein was identified by the determination of its N-terminal amino acid sequence, molecular mass, isoelectric point etc. The results indicated that the primary structure of the expressed protein accorded with the theoretics. Endothelial cell proliferation assay, endothelial cell migration assay and chick chorioallantoic member assay, the bioactivity of vasostatin was investigated. It was proved that vasostatin can inhibit endothelial cell proliferation and migration, and inhibit angiogenesis of chick chorioallantoic member.

Abstract:An apoptosis-related serine protease (ARSP1) was purified from Eisenia fetida extract (mainly a group of anti-tumor protein components) by hydrophobic interaction chromatography and ion exchange chromatography. The molecular mass assayed by SDS-PAGE and isoelectric point of ARSP1 were 28 ku and less than 3.8, separately. However, several coterminous bands could be observed by PAGE of natural ARSP1 and several coterminous peaks of ARSP1 were also detected with MALDI-TOF-MS when the relative molecular mass of three main peaks are 24 645, 25 052 and 25 281, separately. The N-term amino acid sequence of ARSP1 was assayed as following: I(V)IGGT(S)N(D)ASPGEFPWQLSQTRGGSHS. And, a result that ARSP1 is highly homologous with serine proteases was concluded by the comparison of N-term amino acid sequences. In vitro, the cytotoxicity of ARSP1 was not only identified by phase-contrast microscopy observation of apoptotic cells, but also studied further by the localization of fluorescent antibodies. By Schiff's staining, ARSP1 was identified to be glycoprotein or glycopeptide. By fibrin plate assay, ARSP1 was identified to be a plasmin and also a plasminogen activator, and the fibrinolytic activity was inhibited by PMSF (an inhibitor of serine proteases).

Abstract:The obstacle for pig-to-human transplantation is hyperacute rejection (HAR) triggered by the interaction between human natural antibodies and the antigenic epitope Gal-α-1,3-Gal. The α-Gal structure is considered to be the major xenoantigenic epitopes present on porcine tissues. A phage-displayed peptide library is used to identify a 17-amino-acid peptide CCWLLRQPVRFVRSIRS that binds to the mAb anti-B (anti-B monoclonal antibody which binds the carbohydrate Gal α-1,3 Gal).The melibiose competes with the binding of mAB anti-B to the peptide, suggesting that they may bind the same site. Using a pig RBC agglutination assay, it is shown that this peptide can inhibit the agglutination of pig RBCs by human serum or GS-I-B4.

Abstract:To develop a real-time PCR based on TaqMan technology using the new MGB probe for detecting Chlamydia trachomatis DNA，plasmid containing the sequence of interest was constructed for the standardization of the method and to assess its sensitivity. Primers and MGB probe were chosen in the conserved region of cryptic plasmid pLGV440. The Results showed that this Chlamydia trachomatis assay had a threshold sensitivity of one genome copy number per reaction. A linear standard curve was obtained between 10⁰ and 10⁹ DNA copies/reaction (r＞0.990). Fifty clinical specimens were tested by real-time PCR and LCR simultaneously and the coherence was 100%. These observations suggested that real-time PCR based on MGB probe was an excellent candidate for a standard Chlamydia trachomatis detection method in a large scale.

Abstract:A small DNA-binding protein (Ssh10b) from the hyperthermophilic archaeon Sulfolobus shibatae was crystallized. The crystals could only diffract to 0.65 nm. And gel medium was used to improve the diffraction resolution. Gel adding led to slower speed of crystallization, change of crystal morphology and shortening of C axis of unit cell. The crystals grown in gel medium could diffract to 0.35 nm, and a set of data was collected.

Abstract:To establish a rapid construction cRNA standard curves method applicable to common laboratory, PCR was carried to amplify the target gene and internal reference gene using primers containing T7 promotor and polyT sequence, PCR product was cloned as the template of T7 RNase polymerase to synthesize cRNA in vitro for establishing standard curves. The result shows that constructed cRNA curve has a wide linear range of at least six orders and correlation coefficient is 0.99.The rapid and simple method can be applicable to measurement of transcripts for any gene.

Abstract:COX-2 is a multifunctional neuronal modulator, however, its many functions are still unclear and need to be investigated further. Normal adult rat COX-2 cDNA was amplified by PCR from rat brain cDNA library. Sequencing result showed that the cloned gene was identical with that having reported. Expression vectors of whole COX-2 coding gene and its partial coding sequence in carboxyl terminal were respectively constructed by PCR and gene recombination techniques and were transformed into E.coliDH5α, for IPTG-induced expression. The results showed that there was no COX-2 fusion proteins expressed in E.coliDH5α which were transformed with expression vectors of whole COX-2 coding gene, whereas in E.coliDH5α which were transformed with expression vectors of partial coding gene in carboxyl terminal, COX-2 fusion protein was expressed in the form of inclusion body. A protein band of M_r being 44 000 appeared on SDS-PAGE gel. The protein expressed by carboxyl terminal coding sequence had a high purity after denaturation, refolding and purification. New Zealand rabbits were immunized with COX-2 fusion proteins to prepare a polyclonal antibody against COX-2. The COX-2 antiserum was obtained and characterized by ELISA, Western blot, immunohistochemistry and immunocytochemistry. Results showed that the antibody has high titer, affinity and specificity. The studies provide a favorable tool for further functional study of COX-2 in future.

Abstract:Hemagglutinin (HA) gene is an important gene of protective antigen of Avian Influenza Virus (AIV).In order to study HA gene vaccine, the H5 gene of AIV, amplified by PCR, was subcloned into eukaryotic expressing vectors pcDNA4/HisMax and pRc/CMV. The recombinant plasmids were transfected into HeLa cell by Tfx^TM-20 transfection reagent, Superfect transfection reagent and electroporation respectively. The expressed HA was examined and identified by Western blotting and hemagglutination assay. The results showed that HA was expressed precisely in HeLa cells and had bioactivity. Three special bands (HA, HA1 and HA2) were found by Western blotting, which were identical to those of AIV. The quantity of expressed HA from pC4H5,transfected by superfect transfection reagent, was approximately 8 times as much as that from pCMVH5. These results suggest that pC4H5 is a highly efficient eukaryotic expressing vector.