Expression of MLAA34-HSP70 fusion gene constructed by SOE-PCR

To construct the pIRES2-MLAA34-HSP70 recombinant vector and express the MLAA34-HSP70 recombinant proteins in Escherichia coli [E. coli]. The MLAA34 and the HSP70 genes were extracted from U937 cells by RT-PCR, and then we amplified the fusion gene MLAA34-HSP70 by SOE-PCR and inserted it into the pIRES2-EGFP vector to construct the pIRES2-MLAA34-HSP70 recombinant vector. We amplified the fusion gene MLAA34-HSP70 successfully and identified the correctness of pIRES2-MLAA34-HSP70 recombinant vector by PCR and restriction endonuclease. Moreover, the MLAA34-HSP70 recombinant proteins expressed in E. coli were consistent with the expected molecular weight. We constructed the pIRES2-MLAA34-HSP70 recombinant vector successfully and the MLAA34-HSP70 recombinant proteins were successfully expressed by the induction of IPTG

Overexpression of recombinant human teriparatide, rhPTH [1-34] in Escherichia coli: an innovative gene fusion approach

Background: Parathyroid hormone is an 84-amino acid peptide secreted by the parathyroid glands. Its physiological role is maintenance of normal serum calcium level and bone remodeling. Biological activity of this hormone is related to N-terminal 1-34 amino acids. The recombinant form of hormone [1-34] has been approved for treatment of osteoporosis from 2002. In this study, a novel fusion partner has been developed for preparation of high yield recombinant 1-34 amino acids of hPTH

Methods: Novel nucleotide cassette designed encoding a chimeric fusion protein comprising of a fusion partner consisting of a His-tag in N-terminal, 53 amino acids belong to Escherichia coli [E. coli] beta-galactosidase [LacZ] gene, a linker sequence for increasing of expression and protection of target peptide structure from fusion tag effect, an Enteropeptidase cleavage site, rhPTH [1-34] gene fragment. Optimized fusion gene was synthesized and ligated into pET-28a vector under control of T7 promoter, and then transformed in E. coli [DH5 alpha] cells. Positive clones containing this gene were double digested with NcoI and-BamHI and also approved by sequencing. Gene overexpression was observed in SDS-PAGE after induction with 0.2 mM IPTG. Confirmation of gene expression was performed by western blotting using anti-His-tag antibody conjugated with peroxidase

Results: By this fusion gene design approach, we achieved a high level expression of the rhPTH, where it represented at least 43.7% of the total protein as determined by SDS-PAGE and confirmed by western blotting

Conclusion: In addition to high level expression of the designed gene in this work, specific amino acid sequence of bacterial beta-galactosidase was selected as major part of carrier tag for protection of this hormone as important step of recombinant rhPTH with relevant isoelectronic point [pI]. This innovation resulted in recombinant production of hPTH very well and the gene construct could be applied as a pattern for similar recombinant peptides where recombinant protein degradation is a critical issue

Notícias do Levantamento de Recursos e Necessidades de Enfermagem na Revista Brasileira de Enfermagem (1955-1958) / News of the Inquiry about Nursing Needs and Resources in the Brazilian Journal of Nursing (1955-1958) / Noticias del Levantamiento de Necesidades y Recursos de Enfermería en la Revista Brasileña de Enfermería (1955-1958)

Estudo Histórico Social que tem como objeto notícias sobre o Levantamento de Recursos e Necessidades de Enfermagem no Brasil, publicadas na Revista Brasileira de Enfermagem entre 1955 e 1958. A fonte primária foi constituída pelos exemplares da Revista Brasileira de Enfermagem, publicados dentro do recorte temporal do estudo. As fontes secundárias foram constituídas por livros, artigos, dissertações e teses relativas à história da Enfermagem. A análise dos dados teve apoio das fontes secundárias e do pensamento do sociólogo Pierre Bourdieu. Os dados evidenciaram que a Revista Brasileira de Enfermagem, além de oportunizar a divulgação de notícias acerca do Levantamento, proporcionou visibilidade ao mesmo mediante a veiculação dessas notícias e, por fim, teve o efeito simbólico de conferir poder e prestígio à Enfermagem Brasileira.

Social historical study that has as object news related to the Assessment of the Resources and Needs of Nursing in Brazil published in the Revista Brasileira de Enfermagem between 1955 and 1958. The primary source is constituted of copies of Revista Brasileira de Enfermagem published within the selected period of the study. The secondary sources are constituted of books, papers, dissertations and thesis related to the Nursing history. The data analysis was supported by the secondary sources and the thought of the sociologist Pierre Bourdieu. The results evidenced that Revista Brasileira de Enfermagem, in addition to making possible the dissemination of news about the Assessment provided visibility to it and, at last, had the symbolic effect of giving power and prestige to the Brazilian Nursing.

Estudio Histórico Social que tiene como objeto noticias referentes al Levantamiento de Recursos y Necesidades de Enfermería en Brasil publicadas en la Revista Brasileira de Enfermagem entre 1955 y 1958. La fuente primaria se constituye de los ejemplares de la Revista Brasileira de Enfermagem publicados dentro del recorte temporal do estudio. Las fuentes secundarias están constituidas de libros, artículos disertaciones y tesis relativas a la historia de la Enfermería. El análisis de los datos tuvo apoyo de las fuentes secundarias y del pensamiento del Sociólogo Pierre Bourdieu. Los resultados evidencian que la Revista Brasileira de Enfermagem, además de posibilitar la divulgación de noticias acerca del Levantamiento proporcionó visibilidad al mismo mediante la divulgación de esas noticias y, por fin, tuve el efecto simbólico de conferir poder y prestigio a la Enfermería Brasileña.

Construction of nasopharyngeal carcinoma CNE-2 cell lines expressing stable fusion suicide gene CD/UPRT. UL49 / 临床耳鼻咽喉头颈外科杂志

OBJECTIVE@#To construct nasopharyngeal carcinoma CNE-2 cell lines expressing stable fusion suicide gene CD/UPRT. UL49.@*METHOD@#The plasmids of pcDNA3.1 (-)E6. BARF1p. CD/UPRT. UL49 was transfected into CNE-2 cells through lipofectamine, and the transfected CNE-2 cells were selected by G418 and prodrugs for getting the cells expressing fusion CD/UPRT. UL49 gene. The protein produced by the suicide gene was tested by Western-blotting in CNE-2 cells.@*RESULT@#Suicide genes were expressed stably in CNE-2 cells.@*CONCLUSION@#We constructed nasopharyngeal carcinoma cell lines CNE-2 expressing stable suicide gene through lipofectamine.

Construction and tobacco transformation of COR and BBE genes hairpin RNA vector of Papaver somniferum / 药学学报

The gene expressions of codeinone reductase (COR) and berberine bridge enzyme (BBE) in Papaver somniferum were blocked by RNA hairpin of RNA interference (RNAi). The complete sequences of COR and BBE genes were cloned by reverse transcription-polymerase chain reaction (RT-PCR), the results of homology comparison revealed that the cloned COR and BBE genes had high homology with the other gene family members reported in the GenBank. The target sequences of COR and BBE genes were screened in accordance with the design principle of RNAi, a 643 bp fusion gene was obtained by the method of overlapping PCR, then plant expression vector ihpRNA was constructed based on intermediate vector pHANNIBAL and plant expression vector pCEPSPS. With that 78 transgenic plants were obtained through Agrobacterium-mediated and 17 positive plants were screened by PCR, that could initially indicate that the target fragments of COR and BBE gene had been integrated into tobacco genome.

Comparison of three methods in construction fusion gene of influenza A virus Nucleoprotein / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To develop a best method of constructing influenza NP fusion gene containing enhanced green fluorescent protein (EGFP).</p><p><b>METHODS</b>The full-length NP gene of influenza A was amplified by RT-PCR and was inserted into an eukaryotic expression vector pEGFP-N1 in order to construct a fusion gene of pEGFP-N1-NP using three different methods. Method one, NP gene containing restriction endonucleases and pEGFP-N1 were both digested using the same restrict enzymes and ligated, yielding the fusion gene of pEGFP-N1-NP. Method two, NP gene was cloned into pMD19-T Vector to construct a plasmid of pMD19-T-NP. The pMD19-T-NP cloned into pEGFP-N1 to construct the fusion gene of pEGFP-N1-NP; Method three, NP gene containing restriction endonucleases was cloned into pMD19-T Simple Vector to construct a plasmid of pMD19-T-NP. The pMD19-T-NP cloned into pEGFP-N1 to construct the fusion gene of pEGFP-N1-NP.</p><p><b>RESULTS</b>The fusion gene of recombinant eukaryotic expression vector pEGFP-N1-NP was successfully constructed by using method three.</p><p><b>CONCLUSIONS</b>The full-length NP gene is obtained and its fusion gene of recombinant eukaryotic expression plasmid is successfully constructed. This study provides foundation for further understanding the biological function of NP protein and the mechanism of diseases induced by influenza A virus.</p>

Design and expression of an inhibitor for HIV-1 targeting dendritic cell / 生物工程学报

Human immunodeficiency virus (HIV) infects the host cells by the fusion of viral and cell membranes. Blocking the combining between HIV and the receptors can prevent HIV from entering the host cells. We designed an invasion-inhibitor for HIV-1 targeting dendritic cell (DC), including 2 important HIV-1 receptors CD4 and CCR5, and 2 molecules Flt3-L and Mip-3alpha. With the synthetic gene of the inhibitor, 2 eukaryotic expression vectors pABK-CKR5-CD4/Flt3L-Mip3alpha (pABK-HIV-MF) and pABK-CKR5-CD4 (pABK-HIV-MT) were constructed and transfected into HEK 293 cells for expression. Results from RT-PCR, immunofluorescent assay, ELISA and Western blot approved that the invasion-inhibitor for HIV-1 was successfully and exactly expressed in the eukaryotic cells. Current study formed a solid base for the further research on the function of inhibitors for HIV-1 and elimination targeting DC.

Construction and immunogenicity of recombinant porcine parvovirus-like particles with somatostatin / 生物工程学报

In order to obtain a virus-like particle vaccine both for porcine parvovirus (PPV) prevention and growth-promotion, VP2 gene of PPV NJ-a strain was amplified with PCR, and four copies of synthetic somatostatin gene were fused to the N-terminal of VP2 gene. The fused gene was cloned into pFast-HT A to construct the recombinant plasmid pFast-SS4-VP2, then the pFast-SS4-VP2 was transformed into DH10Bac competent cells and recombined with shuttle vector Bacmid, followed by identification with blue-white screening and PCR analysis for three cycles, and the positive recombinant was named as rBacmid-SS4-VP2. The positive Sf-9 cells were transfected with rBacmid-SS4-VP2 by Lipofectamine to produce recombinant baculovirus. When the cytopathic effect (CPE) was obvious, the transfected Sf-9 cell was harvested, and the positive recombinant virus was named as rBac-SS4-VP2. The insertion for the target gene into baculovirus genome was confirmed with PCR. SDS-PAGE and Western blotting revealed that the calculated protein of approximately 68 kDa was in the expressed in the insect cells. The Sf-9 cells infected with rBac-SS4-VP2 were stained positive against PPV antibody using the indirect immunofluorescence assay (IFA). Moreover, the virus particle self-assembly was observed under electron microscopy. 90 four-week-old mice were immunized by the recombinant protein coupled with different adjuvants alhydrogel, IMS and oil. VP2-specific ELISA antibodies, PPV-specific neutralizing antibody, somatostatin antibody and growth hormone levels were examined to evaluate the immunogenicity of this virus like particle. Results indicated that mice groups immunized rSS4-VP2 protein with alhydrogel and IMS developed similar humoral immune response comparing with inactived PPV vaccine. Mice group immunized with rSS4-VP2 generated higher level of SS antibody and growth hormone comparing with negative control, mice receiving rSS4-VP2 with alhydrogel developed the highest antibody titre than all other groups, while the oil group developed the lowest antibody level. This study provides not only a new rout for production of safe and effective virus like particle subunit vaccine, but also the foundations for peptide presentation and multivalent subunit vaccine design.

Transcriptional Activity of Plasmodium Subtilisin-like Protease 2 (Pf-Sub2) 5'Untranslated Regions and Its Interaction with Hepatocyte Growth Factor

The onset, severity, and ultimate outcome of malaria infection are influenced by parasite-expressed virulence factors and individual host responses to these determinants. In both humans and mice, liver injury is involved after parasite entry, which persists until the erythrocyte stage after infection with the fatal strain Plasmodium falciparum (Pf). Hepatocyte growth factor (HGF) has strong anti-apoptotic effects in various kinds of cells, and also has diverse metabolic functions. In this work, Pf-subtilisin-like protease 2 (Pf-Sub2) 5'untranslated region (UTR) was analyzed and its transcriptional activity was estimated by luciferase expression. Fourteen TATA boxes were observed but only one Oct-1 and c-Myb were done. In addition, host HGF interaction with Pf-Sub2 was evaluated by co-transfection of HGF- and Pf-Sub2-cloned vector. Interestingly, -1,422/+12 UTR exhibited the strongest luciferase activity but -329 to +12 UTR did not exhibit luciferase activity. Moreover, as compared with the control of unexpressed HGF, the HGF protein suppressed luciferase expression driven by the 5'untranslated region of the Pf-Sub2 promoter. Taken together, it is suggested that HGF controls and interacts with the promoter region of the Pf-Sub2 gene.

Construction and characterization of a novel somatostatin prokaryotic expression / 生物工程学报

In the current work, the fusion gene including somatostatin (SS) and the hepatitis B surface antigen gene was cloned into a balanced lethal system plasmid (pYA3493), and then transformed into asd- attenuated Salmonella choleraesuis C500 strain, the positive transformant without antibiotic resistance gene was confirmed by restriction analysis and DNA sequencing, designated as pYA-SS. The expression and immunogenicity of fusion protein were detected by SDS-PAGE and Western blot analysis. These results show that the recombinant prokaryotic expression plasmid pYA-SS could express the SS fusion protein with good immunogenicity in C500 strain. In above all, this study could provide reliable materials to develop novel, good and safe vaccine in enhancing the growth of animals.

Expression of BMP-2/7 heterodimers with potent ability to stimulate osteogenic differentiation in CHO cells / 生物工程学报

Coding sequences of BMP-2 and BMP-7 were amplified using PCR and ligated with a DNA sequence encoding a flexible peptide (Gly4Ser)5. The fusion gene was inserted into plasmid pIRESneo3. The expression level of BMP2/7 heterodimers in the transfected CHO-K1 cells was 230.75+/-13.34 ng/mL. Culture medium of stably tansfected clone pool was collected as conditional medium to treat osteoblast MC3T3 cells. Staining of Alkalin phosphatase and Alizarin red demonstrated that the conditional medium significantly promoted osteogenic differentiation to a higher extent than BMP-2 homodimers expressed in either CHO-K1 cells or E. coli. Transcriptional levels of Osteogenic phenotype-related molecular markers such as OC, ALP, Runx2 and Osx were increased (P<0.05), and BMP/Smad signal activities were significantly enhanced by BMP-2/7 heterodimers, comparing with BMP-2 homodimers (P<0.05). The results demonstrate that BMP-2/7 heterodimers expressed in CHO-K1 cells have potent ability to stimulate osteogenic differentiation.

An efficient tool for the construction of multiple-cistronic vectors: FMDV 2A / 生物工程学报

Recently, cancer therapy with mutiple genes has been attached with great attention. However, at present there is no efficient tool to construct multiple-cistrons. The large sizes and the imbalance in expression of most traditional tools, such as ribosome entry sites (IRESes),greatly block their wide employment in the construction of multiple cistronic gene therapy vectors. The self-cleaving peptide 2A from foot-and-mouth disease virus (FMDV) has a very small size, and more importantly, high cleavage activity in artifical bicistron, which bring new hope for mutiple genes therapy stategy. In this article, the characteristics and cleavage activities of FMDV 2A will be elucidated,and we further outline its applications in cancer gene therapy.

Mice transduced with double-mutant dihydrofolate reductase-cytidine deaminase fusion gene attained protection from high dose chemotherapy / 中华外科杂志

<p><b>OBJECTIVE</b>To explore the feasibility of transferring fusion gene of dihydrofolate reductase (DHFR) gene and cytidine deaminase (CD) gene into mouse bone marrow cells in order to observe the drug resistance of high dose methotrexate (MTX) and cytosine arabinoside (Ara-C) in the bone marrow cells and to improve the tolerance of myelosuppression following combination chemotherapy.</p><p><b>METHODS</b>Human double-mutant dihydrofolate reductase-cytidine deaminase fusion gene was transferred into two mice bone marrow cells by retroviral vector. Resistant colony-forming unit granulocyte-macrophage (CFU-GM) assays were performed in mouse bone marrow cells by retroviral infection and after treatment by drugs (Ara-C, MTX, and Ara-C + MTX). DNA was extracted from mouse bone marrow cells. The expression of drug resistant genes in mouse bone marrow cells after transferring by retroviral vector was checked by polymerase chain reaction (PCR).</p><p><b>RESULTS</b>Bone marrow cells after coculture with the retroviral producer cells transduced with the genes (SFG-F/S-CD) showed the drug resistance colonies yield (Colony formation after exposure to Ara-C, MTX and Ara-C + MTX were 56%, 22% and 14%, respectively) and the increase in drug resistant to both MTX and Ara-C (P < 0.005). Expression of DHFR and CD gene in extracted DNA of transfected mice were demonstrated by PCR.</p><p><b>CONCLUSIONS</b>Double drug resistant gene can not only integrate and co-express in mice bone marrow cells but also increase the drug resistance to MTX and Ara-C.</p>

Cloning of CTB-PROIN fusion gene and its expression in Escherichia coli / 生物工程学报

A fusion gene CTB-PROIN, in which Proinsulin gene was fused to the 3' end of CTB gene by a hinge peptide 'GPGP', was constructed and cloned into pET-30a(+) to obtain a prokaryotic expression vector pETCPI. Subsequently the recombinant plasmid pETCPI was transformed into E. coli stain BL21 (DE3). After induced by IPTG, the expression product was analyzed by sodium dodecyl sulphate-polyacrylamide gel (15%) electrophoresis (SDS-PAGE), and its result indicated that the recombinant protein CTB-PROIN was expressed and accumulated as inclusion bodies. The recombinant CTB-PROIN protein accumulated to the level of 25% of total bacterial proteins. After inclusion bodies was denaturalized and refolded in vitro, significant assembly of monomers had occurred, and the recombinant protein represented assembled pentamers. The results of western blotting analysis also demonstrated that the fusion protein could be recognized by the anti-CT and anti-insulin antibody, respectively. In addition, the result of the CTB-PROIN-GM1 binding assay, that the protein could bind to monosialoganglioside specifically, showed it possesed biological activity in vitro. These results provided the possibility of developing a cheaper and more efficient oral vaccine for type I diabetes using such constructs.

Immunogenicity of DNA vaccine expressing GP5 of porcine reproductive and respiratory syndrome virus fused with VP22 of bovine herpesvirus 1 / 生物工程学报

To enhance the immuogenicity of DNA vaccines expressing the GP5 protein of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), the tegument protein VP22 (encoded by VP22 gene) of Bovine Herpesvirus 1 (BHV-1), which has been demonstrated to exhibit the unusual protein transduction property, was fused to N-terminus of GP5 of DNA vaccine construct pCI-ORF5M, resulting in pCI-VP22-ORF5M expressing VP22-GP5 fusion protein. The expression of VP22-GP5 fusion protein was confirmed by both indirect immunofluorescence assay (IFA) and Western blot. To investigate its immunogenicity, BALB/c mice were immunized with the fusion expression plasmid pCI-VP22-ORF5M and non-fusion expression plasmid pCI-ORF5M, respectively. The GP5-specific ELISA antibodies, neutralizing antibodies and lymphocyte proliferative responses were evaluated at various time points after primary immunization. The results showed that GP5-specific ELISA antibodies, neutralizing antibodies, and lymphocyte proliferative responses induced by DNA vaccine pCI-VP22-ORF5M were higher significantly than those of DNA vaccine pCI-ORF5M, indicating that fusion expression with BHV-1 VP22 significantly enhances the immuogenicity of DNA vaccine expressing the PRRSV GP5 protein, and that this strategy may also be useful to develop more efficient DNA vaccines against other pathogens.

Expression of fusion protein of parathyroid hormone and transferrin N-terminal half-molecule in Pichia pastoris / 生物工程学报

The fused gene (PTH-TFN) of parathyroid hormone (PTH) gene and transferring N-terminal half-molecule (TFN) gene was amplified by multiple PCR and inserted into pPIC9 vector. The recombinant plasmid pPIC9-PTH-TFN was transformed into Pichia pastoris GS115 by PEG. After methanol induction, the target protein was expressed in fermentation supernatant at high level. The fused protein PTH-TFN with purity being higher than 95% was finally obtained after purification through two-step chromatography: SP Sepharose Fast Flow and Phenyl Sepharose Fast Flow. Western blot analysis and adenylate cyclase assay proved that the fused protein exhibited the bioactivity to stimulate cAMP synthesis and the ability to bind Fe3+ in the Fe3+ saturation study as the recombinant TFN did indicating that TFN could be used as the transcellar carrier of PTH.

Chromosomal changes detected by fluorescence in situ hybridization in patients with acute lymphoblastic leukemia / 中华医学杂志(英文版)

<p><b>OBJECTIVES</b>To investigate patients with acute lymphoblastic leukemia (ALL) for TEL/AML1 fusion, BCR/ABL fusion, MLL gene rearrangements, and numerical changes of chromosomes 4, 10, 17 and 21 by fluorescence in situ hybridization (FISH) and to determine the relationship and the significance of those findings.</p><p><b>METHODS</b>Fifty-one American patients (34 men and 17 women) were included in this study. Of them there were 41 patients with pro-B cell type ALL, 9 with B cell type ALL and 1 with T cell type ALL. Chromosome metaphases of each sample were prepared according to standard protocols. Fluorescence in situ hybridization was performed using commercially available DNA probes, including whole chromosome painting probes, locus specific probes, specific chromosome centromere probes and dual color/multiple color translocation fusion probes. The digital image analysis was carried out using Cytovision and Quips FISH programs.</p><p><b>RESULTS</b>An overall incidence of chromosomal anomalies, including t (9;22), MLL gene rearrangements, t (12;21), and numerical chromosomal anomalies of chromosomes 4, 10, 17 and 21 was found in 33 patients (65%). Thirty-one of them were pediatric patients and two adults. The t (12;21) was the commonest chromosomal anomaly detected in this population; 14 out of the 45 pediatric patients (31%) were positive for TEL/AML1 fusion, among which three had an additional derivative 21 [t (12;21)], four had a deletion of 12p and two had an extra copy of chromosome 21. All 14 patients with positive TEL/AML1 fusion had ALL pre-B cell or B-cell lineage according to standard immunotyping. The percentage of cells with fusion signals ranged from 20% to 80%. All fourteen patients positive for TEL/AML1 gene fusion were mosaic. Three out of the 14 patients positive for the TEL/AML1 gene fusion were originally reported to be culture failures and none of the remaining eleven samples had been found to have chromosome 12 abnormalities by conventional cytogenetic techniques. All pediatric patients with pre-T or T cell lineage and the six adults were negative for TEL/AML1 fusion. One patient had double Philadelphia chromosomes, three had a rearrangement or a deletion of the MLL gene, one had t (4;11) and two had a deletion of the MLL. One of the patients with an MLL deletion also had a large ring of chromosome 21, and r (21) was caused by AML1 gene tandemly duplicated at least five times. The second case with the MLL deletion was also unique, the patient had a t (12;21) as well. A total of 20 patients had numerical changes (gain or loss) of chromosomes 4, 10, 17 and 21. Eight patients were found to have trisomies of three or four different chromosomes. Interestingly, seven of these patients did not have TEL/AML1, BCR/ABL or the MLL gene rearrangement; one did have the TEL/AML1 gene fusion. Eleven patients with pro-B cell or B cell type ALL (9 children with ALL, 2 adults with ALL) had numerical changes of chromosome 21 (gain 1 or 2 chromosome 21), among them, 10 patients had no structural alteration of chromosome 21, and one was combined by t (12; 21). Four patients had a monosomy of chromosome 17 and three out of these patients with monosomy 17 also had a fusion signal of TEL/AML1.</p><p><b>CONCLUSIONS</b>FISH plays an important role in detecting chromosome changes, especially in some cryptic chromosome translocations and patients with culture failures. This study found a trend towards a division between patients who had structural changes such as t (12;21) or a ring chromosome 21 and those who had numerical changes of chromosome 21 as well as the patients with TEL/AML1 fusion and patients with the coexistence of numerical chromosomal changes of chromosomes 4, 10 and 17. In our opinion there are two separate mechanisms which lead to the development or progression of leukemia.</p>

Construction and expression of somatostatin (S14) and hepatitis B surface antigen gene in yeast Pichia pastoris / 生物医学工程学杂志

To construct the ss/HBsAg protein gene-engineering vaccine for developing the diagnosis and cure tumors in clinical medicine and promoting the growth in animal husbandry production. A pair of primers were designed according separately to the sequence of Somatostatin gene(S14) and HBsAg gene. Their gene fragments were separately amplified by using PCR and cloned, following sequencing, the DNA fragments were inserted into pBluescript vector. Then the ss/HBsAg chimera was constructed and was cloned into pPICZaA plasmid, and transformed into electroporated Pichia pastoris. High yield protein expression was obtained. Expressed protein was proved with high specificity and it's molecular weigh was about 28 KD identified by SDS-PAGE and Western blot.

5'NCR Incipient establishment of HCV 5'NCR transgenic mice model / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To evaluate the inhibitory effects of HCV antisense oligonucleotide drugs in vivo.</p><p><b>METHODS</b>Transgenic mice were generated by microinjection. The construct of luciferase controlled by HCV 5'NCR that contains CMV promotor was injected into the male pronuclus of fertilized eggs of ICR mice.</p><p><b>RESULTS</b>Sixty-eight survival birth transgenic mice were identified by PCR amplification with tail DNA, 13 of whom were positive with an integration ratio of 19.2% (13/68). Transgenic mRNA was detected by RT-PCR in the tissue of three mice's offspring that contain transgenic DNA. Luciferase expression was detected in a line (35#) using the luciferase assay system and the expression persisted in the F2 generation. The phenotype of the mice in this line was normal and there was no significant difference in physiology from normal mice.</p><p><b>CONCLUSIONS</b>This line of transgenic mice will provide a useful animal model for the study of function of HCV 5'NCR and the evaluation of HCV antisense drugs in vivo.</p>