Prokaryotic expression of a recombinant protein of adeno-associated virus capsid conserved regions and preparation of its polyclonal antibody / 南方医科大学学报

OBJECTIVE@#To express and purify the antigenic peptide of adeno-associated virus (AAV) capsid conserved regions in prokaryotic cells and prepare its rabbit polyclonal antibody.@*METHODS@#The DNA sequence encoding the conserved regions of AAV capsid protein was synthesized and cloned into the vector pET30a to obtain the plasmid pET30a-AAV-CR for prokaryotic expression and purification of the conserved peptides. Coomassie blue staining and Western blotting were used to identify the AAV conserved peptides. Japanese big ear white rabbits were immunized with AAV conserved region protein to prepare polyclonal antibody, with the rabbits injected with PBS as the control group. The antibody titer was determined with ELISA, and the performance of the antibody for recognizing capsid protein sequences of AAV1-AAV10 was assessed with Western blotting and immunofluorescence assay.@*RESULTS@#The plasmid pET30a-AAV-CR was successfully constructed, and a recombinant protein with a relative molecular mass of 17000 was obtained. The purified protein induced the production of antibodies against the conserved regions of AAV capsid in rabbits, and the titer of the purified antibodies reached 1:320 000. The antibodies were capable of recognizing a wide range of capsid protein sequences of AAV1-AAV10.@*CONCLUSION@#We successfully obtained the polyclonal antibodies against AAV capsid conserved region protein from rabbits, which facilitate future studies of AAV vector development and the biological functions of AAV.

Selective intra-dinucleotide interactions and periodicities of bases separated by K sites: a new vision and tool for phylogeny analyses

Direct tests of the random or non-random distribution of nucleotides on genomes have been devised to test the hypothesis of neutral, nearly-neutral or selective evolution. These tests are based on the direct base distribution and are independent of the functional (coding or non-coding) or structural (repeated or unique sequences) properties of the DNA. The first approach described the longitudinal distribution of bases in tandem repeats under the Bose-Einstein statistics. A huge deviation from randomness was found. A second approach was the study of the base distribution within dinucleotides whose bases were separated by 0, 1, 2... K nucleotides. Again an enormous difference from the random distribution was found with significances out of tables and programs. These test values were periodical and included the 16 dinucleotides. For example a high ¨positive¨ (more observed than expected dinucleotides) value, found in dinucleotides whose bases were separated by (3K + 2) sites, was preceded by two smaller ¨negative¨ (less observed than expected dinucleotides) values, whose bases were separated by (3K) or (3K + 1) sites. We examined mtDNAs, prokaryote genomes and some eukaryote chromosomes and found that the significant non-random interactions and periodicities were present up to 1000 or more sites of base separation and in human chromosome 21 until separations of more than 10 millions sites. Each nucleotide has its own significant value of its distance to neutrality; this yields 16 hierarchical significances. A three dimensional table with the number of sites of separation between the bases and the 16 significances (the third dimension is the dinucleotide, individual or taxon involved) gives directly an evolutionary state of the analyzed genome that can be used to obtain phylogenies. An example is provided.

Progress in proteogenomics of prokaryotes / 生物工程学报

With the rapid development of genome sequencing technologies, a large amount of prokaryote genomes have been sequenced in recent years. To further investigate the models and functions of genomes, the algorithms for genome annotations based on the sequence and homology features have been widely implemented to newly sequenced genomes. However, gene annotations only using the genomic information are prone to errors, such as the incorrect N-terminals and pseudogenes. It is even harder to provide reasonable annotating results in the case of the poor genome sequencing results. The transcriptomics based on the technologies such as microarray and RNA-seq and the proteomics based on the MS/MS have been used widely to identify the gene products with high throughput and high sensitivity, providing the powerful tools for the verification and correction of annotated genome. Compared with transcriptomics, proteomics can generate the protein list for the expressed genes in the samples or cells without any confusion of the non-coding RNA, leading the proteogenomics an important basis for the genome annotations in prokaryotes. In this paper, we first described the traditional genome annotation algorithms and pointed out the shortcomings. Then we summarized the advantages of proteomics in the genome annotations and reviewed the progress of proteogenomics in prokaryotes. Finally we discussed the challenges and strategies in the data analyses and potential solutions for the developments of proteogenomics.

Prokaryotic expression of a truncated form of bovine herpesvirus 1 glycoprotein E (gE) and its use in an ELISA for gE antibodies / Expressão procariota de uma forma truncada da glicoproteína E (gE) do herpesvírus bovino tipo 1 e uso em ELISA para anticorpos contra a gE

This article describes the expression of a truncated form of bovine herpesvirus 1 (BoHV-1) glycoprotein E (gE) for use as immunodiagnostic reagent. A 651 nucleotide fragment corresponding to the amino-terminal third (217 amino acids) of BoHV-1 gE - that shares a high identity with the homologous BoHV-5 counterpart - was cloned as a 6×His-tag fusion protein in an Escherichia coli expression vector. A soluble protein of approximately 25 kDa purified from lysates of transformed E. coli was recognized in Western blot (WB) by anti-6xHis-tag and anti-BoHV-1 gE monoclonal antibodies. In addition, the recombinant protein was specifically recognized in WB by antibodies present in the sera of cattle seropositive to BoHV-1 and BoHV-5. An indirect ELISA using the expressed protein as coating antigen performed comparably to a commercial anti-gE ELISA and was able to differentiate serologically calves vaccinated with a gE-deleted BoHV-5 strain from calves infected with BoHV-1. Thus, the truncated gE may be useful for serological tests designed to differentiate BoHV-1/BoHV-5 infected animals from those vaccinated with gE-negative marker vaccines.

Este trabalho relata a expressão de uma forma truncada da glicoproteína E (gE) do herpesvírus bovino tipo 1 (BoHV-1) para uso em imunodiagnóstico. Um fragmento de 651 pares de bases (pb) correspondente ao terço amino-terminal (217 aminoácidos) da gE do BoHV-1 - que compartilha uma alta identidade com a gE do BoHV-5 - foi clonada como proteína de fusão com cauda 6x de histidina em um vetor de expressão em Escherichia coli. Uma proteína solúvel de aproximadamente 25 kDa purificada de lisados de E.coli foi reconhecida em Western blot (WB) por anticorpos monoclonais anti-6xHis-tag e anti-gE. Além disso, a proteína recombinante purificada foi reconhecida em WB por anticorpos presentes no soro de animais soropositivos ao BoHV-1 e BoHV-5. Um ELISA indireto utilizando a proteína recombinante como antígeno apresentou performance comparável a um ELISA gE comercial e foi capaz e diferenciar sorologicamente animais vacinados com uma cepa gE-negativa de BoHV-5 de animais infectados com o BoHV-1. Portanto, a gE truncada pode ser útil em testes sorológicos diferenciais para uso conjunto com vacinas com marcador antigênico gE para o BoHV-1 e BoHV-5.

Prokaryotic cells: structural organisation of the cytoskeleton and organelles

For many years, prokaryotic cells were distinguished from eukaryotic cells based on the simplicity of their cytoplasm, in which the presence of organelles and cytoskeletal structures had not been discovered. Based on current knowledge, this review describes the complex components of the prokaryotic cell cytoskeleton, including (i) tubulin homologues composed of FtsZ, BtuA, BtuB and several associated proteins, which play a fundamental role in cell division, (ii) actin-like homologues, such as MreB and Mb1, which are involved in controlling cell width and cell length, and (iii) intermediate filament homologues, including crescentin and CfpA, which localise on the concave side of a bacterium and along its inner curvature and associate with its membrane. Some prokaryotes exhibit specialised membrane-bound organelles in the cytoplasm, such as magnetosomes and acidocalcisomes, as well as protein complexes, such as carboxysomes. This review also examines recent data on the presence of nanotubes, which are structures that are well characterised in mammalian cells that allow direct contact and communication between cells.

[ comparison of bone morphogenetic protein-7 mutant expression in prokaryotic and eukaryotic hosts]

Bone morphogenetic protein-7 [BMP-7] is a multifunctional growth factor predominantly recognized for its osteoinductive properties. Due to the high cost of this protein, the availability of BMP-7 for treatment is limited. The heterologous production of recombinant hBMP-7 has been performed in a number of expression systems. In this study a novel form of BMP-7 was expressed in eukaryotic and prokaryotic hosts. For expression in the prokaryotic system, the novel protein was secreted to the periplasmic space of Escherichia coli using a pelB signal sequence followed by singlestep purification by Ni2+-charged column chromatography. In the mammalian cell expression system, we transferred a full-length cDNA encoding precursor of the novel protein to CHO cells then selected stable clones by using the appropriate antibiotic concentration. Expressions in both systems were confirmed by Western blot analysis. The novel recombinant protein was produced as a 36-38 kDa dimer in the CHO cell line and a 16 kDa monomer in the Escherichia coli system. Quantitative analysis according to ELISA showed that the expression levels of the mutant protein in the eukaryotic and prokaryotic expression systems were 40 ng/ml and 135 ng/ml of the culture media, respectively. In this study, the expression level in Escherichia coli was at least three times more than observed in the CHO cells. However, further optimization is required to obtain a dimer protein in Escherichia coli. The results show that periplasmic expression may be suitable for the production of complex proteins such as BMPs

Automatic prediction of non-coding RNA genes in prokaryotes based on compositional statistics.

Although non-coding RNA (ncRNA) genes do not encode proteins, they play vital roles in cells by producing functionally important RNAs. In this paper, we present a novel method for predicting ncRNA genes based on compositional features extracted directly from gene sequences. Our method consists of two Support Vector Machine (SVM) models — Codon model which uses codon usage features derived from ncRNA genes and protein-coding genes and Kmer model which utilizes features of nucleotide and dinucleotide frequency extracted respectively from ncRNA genes and randomly chosen genome sequences. The 10-fold cross-validation accuracy for the two models is found to be 92% and 91%, respectively. Thus, we could make an automatic prediction of ncRNA genes in one genome without manual filtration of protein-coding genes. After applying our method in Sulfolobus solfataricus genome, 25 prediction results have been generated according to 25 cut-off pairs. We have also applied the approach in E. coli and found our results comparable to those of previous studies. In general, our method enables automatic identification of ncRNA genes in newly sequenced prokaryotic genomes. Datasets and program code used in this work are available at http://cobi.uestc.edu.cn/resource/SS_ncRNA/

Genômica comparativa de procariotos: análise da variabilidade em funções enzimáticas / Comparative genomics of prokaryotes: analysis of variability in enzymatic functions

Esta tese trata de abordagens computacionais para a análise comparativa de genomas em larga escala e da análise da variabilidade em funções enzimática de procariotos. Este trabalho apresenta um banco de dados denominado ProteinWorldDB, que representa um esforço importante para criar um conjunto de dados consistente e confiável de comparações entre o conteúdo protéico codificado por centenas de genomas completamente sequenciados, usando uma abordagem rigorosa baseada em programação dinâmica. Além disto, este trabalho descreve uma metodologia aprimorada para detecção e anotação de pseudogenes em procariotos (e outros organismos com organização genômica similar), e uma análise da ocorrência, distribuição e padrões de extinção de enzimas análogas preditas em procariotos. A base de dados ProteinWorldDB oferece à comunidade científica a oportunidade de minerar dados comparativos calculados de forma precisa e usar a informação disponível – e.g. índices de similaridade (e suas estimativas estatísticas) entre pares ou grupos de proteínas, proteínas ortólogas e parálogas inferidas, genes taxonomicamente restritos (únicos), entre outras – como ponto de partida para análises subsequentes. Nossa metodologia para a detecção e anotação de pseudogenes em procariotos, baseada em comparações entre sequências codificantes e regiões intergênicas de genomas-alvos, apresenta duas inovações importantes: a reconstrução da sequência remanescente a partir de todos os fragmentos encontrados, não somente a partir do mais similar e a determinação de limiares de similaridade adequados ao conjunto de dados analisados, com base em validações estatísticas. A aplicação deste método na busca de pseudogenes na via glicolítica/gliconeogênese de centenas de procariotos resultou em um número expressivo de novos pseudogenes identificados, mostrando a necessidade de se incluir mecanismos de busca sistemática de pseudogenes nos fluxos de anotação genômica de procariotos. A análise da ocorrência, distribuição e padrões de extinção de enzimas análogas preditas na via gicolítica/gliconeogênese de centenas de procariotos nos revelou um quadro complexo, difícil de ser interpretado, mesmo quando apenas um pequeno grupo de espécies selecionadas foi utilizado. Um estudo mais detalhado, relacionando os resultados obtidos ao estilo de vida, filogenia, estrutura e organização genômicas destas espécies, será necessária para tentar responder às questões fundamentais que nos colocamos: como surgem as enzimas análogas e, sobretudo, por que, aparentemente, ocorreram tantos eventos de origem independente de atividades enzimáticas durante a evolução, e por que existem diferentes formas análogas coexistindo no mesmo organismo? A manutenção de duas ou mais formas análogas poderia proporcionar flexibilidade metabólica e, portanto, uma vantagem seletiva, dependendo do ambiente e estilo de vida do organismo; ou, ainda, distintas formas poderiam competir pela execução de uma mesma função e, neste caso, sendo uma das formas mais competitivas que a outra, a desfuncionalização da forma menos competitiva poderia representar uma vantagem seletiva, já que o gasto energético com a biossíntese da enzima seria eliminado. Seria possível considerar enzimas análogas como indivíduos e seus grupos como populações, todos em competição por um nicho metabólico em particular? Neste caso, seria plausível imaginar que enzimas mais competitivas teriam uma vantagem seletiva sobre formas alterativas menos competitvas e, consequentemente, dispensar-se-iam entre os diversos genomas bacterianos, com o passar do tempo.

Cloning and expression of human vascular endothelial growth factor gene and inhibition of its expression by antisense in prokaryotic system

Angiogenesis is an important process in physiology and disease pathogenesis and is controlled in a healthy body by a number of stimulatory and inhibitory factors. The aim of this study was to determine the effect of antisense transcript on the sense transcript of the endothelial growth factor [EGF] gene in bacterial system as an approach for the gene regulation in tumors. The hepatoma cell line [HepG2] was stimulated by PMA. VEGF mRNA was used for RT-PCR. VEGF cDNA was synthesised and cloned into T- vector pTZ57R, then sense fragment of VEGF subcloned into pACYC Duet-1 expression vector and antisense VEGF subcloned into pCDNAS expression vector. Recombinant plasmids were transforemed into BL2 1 bacterial cells. Expression of recombinant plasmid was analysed by western blot technique. The recombinant pCDNA3-VEGF [pYZantiVEGF] was successfully expressed in BL21 cells. Western blot analysis showed that the expression of VEGF decreased significantly in the cells transfected with VEGF antisense RNA compared with the pACYCDUET-1 - VEGF [pYZsenseVEGF] transfected and control. The expression of VEGF in BL21 cells was strong. In vitro, antisense of VEGF inhibited VEGF expression significantly in BL21 cells

Ovine Follistatin gene expression and functional analysis of follistatin domains / 生物工程学报

In order to study ovine follistatin function, we amplified the total of 1038 base pair of ovine complete follistatin cDNA and cloned into pGEM-T vector by RT-PCR from ovine ovary RNA. After removal of the signal peptide it was subcloned into the pET41a to construct the prokaryotic expression vector, named pFSsig-. SDS-PAGE and Western blotting identified the 66 kDa product of the expression of follistatin cDNA. Based on the complete CDS sequence, we cloned follistatin N-terminal domain and domain 1 with PCR and inserted into pLEX-MCS lentiviral vector, named pFS-N+D1. After package and passage of lentivirus in 293T cells, and then infected sheep primary muscle cells (SPMC). The expression of FS N+D1 in SPMC was assayed by Western blotting. The cell growth curve of the infected SPMC and noninfected control cells displayed that FS N+D1 stablly transfected SPMC proliferated significantly faster than the control cells (P < 0.01). Our data inferred that ovine FS N+D1 domain had the function to stimulate sheep muscle cell growth.

Screening and application of prokaryotic enhancer-like sequence 3A / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To screen enhancer-like sequences from Escherichia coli strain C600 genome, to construct an expression vector harboring prokaryotic enhancer-like sequence and study the effect of interferon gene expression.</p><p><b>METHODS</b>Enhancer-like element from Escherichia coli strain C600 genome was obtained by using the chloramphenicol acetyl-transferase (CAT) gene as reporter gene. An expression vector harboring prokaryotic enhancer-like sequence from Escherichia coli strain C600 was constructed. Interferon was expressed and assayed.</p><p><b>RESULTS</b>An enhancer-like sequences with distance and orientation independence property were screened and named 3A. Quantification test showed that the direct and reverse orientation of 3A could increase the activity of beta-galactosidase with 7.11 and 2.93 times. The enhancing activity of the element was on transcription level. An expression vector harboring the prokaryotic enhancer-like sequence 3P3 which was enhancing function region of sequence 3A was constructed. Using this vector the antiviral activity of interferon alpha-2b was increased by 3.7 times in comparison with the original expression plasmid.</p><p><b>CONCLUSION</b>3A enhancer-like sequence was screened from Escherichia coli strain C600 genome. Interferon gene was highly expressed by using an expression vector harboring enhancer-like sequences.</p>

Prokaryotic expression of a novel mouse pro-apoptosis protein PNAS-4 and application of its polyclonal antibodies

Mouse PNAS-4 (mPNAS-4) has 96 percent identity with human PNAS-4 (hPNAS-4) in primary sequence and has been reported to be involved in the apoptotic response to DNA damage. However, there have been no studies reported of the biological functions of mPNAS-4. In studies conducted by our group (unpublished data), it was interesting to note that overexpression of mPNAS-4 promoted apoptotic death in Lewis lung carcinoma cells (LL2) and colon carcinoma cells (CT26) of mice both in vitro and in vivo. In our studies, mPNAS-4 was cloned into the pGEX-6P-1 vector with GST tag at N-terminal in Escherichia coli strain BL21(DE3). The soluble and insoluble expression of recombinant protein mPNAS-4 (rmPNAS-4) was temperature-dependent. The majority of rmPNAS-4 was insoluble at 37°C, while it was almost exclusively expressed in soluble form at 20°C. The soluble rmPNAS-4 was purified by one-step affinity purification, using a glutathione Sepharose 4B column. The rmPNAS-4 protein was further identified by electrospray ionization-mass spectrometry analysis. The search parameters of the parent and fragment mass error tolerance were set at 0.1 and 0.05 kDa, respectively, and the sequence coverage of search result was 28 percent. The purified rmPNAS-4 was further used as immunogen to raise polyclonal antibodies in New Zealand white rabbit, which were suitable to detect both the recombinant and the endogenous mPNAS-4 in mouse brain tissue and LL2 cells after immunoblotting and/or immunostaining. The purified rmPNAS-4 and our prepared anti-mPNAS-4 polyclonal antibodies may provide useful tools for future biological function studies for mPNAS.

Intrinsically bent DNA in replication origins and gene promoters

Intrinsically bent DNA is an alternative conformation of the DNA molecule caused by the presence of dA/dT tracts, 2 to 6 bp long, in a helical turn phase DNA or with multiple intervals of 10 to 11 bp. Other than flexibility, intrinsic bending sites induce DNA curvature in particular chromosome regions such as replication origins and promoters. Intrinsically bent DNA sites are important in initiating DNA replication, and are sometimes found near to regions associated with the nuclear matrix. Many methods have been developed to localize bent sites, for example, circular permutation, computational analysis, and atomic force microscopy. This review discusses intrinsically bent DNA sites associated with replication origins and gene promoter regions in prokaryote and eukaryote cells. We also describe methods for identifying bent DNA sites for circular permutation and computational analysis.

Construction and expression of a prokaryotic vector of recombinant human adiponectin global domain / 南方医科大学学报

<p><b>OBJECTIVE</b>To construct and express the recombinant human adiponectin (gAd) global domain.</p><p><b>METHODS</b>gAd complementary DNA (cDNA) was obtained from human fat tissue by RT-PCR. The PCR product was cloned into the vector pMD18-T and the prokaryotic expression vector pET32a(+). The recombinant vector was identified by digestion with double restriction endonucleases SalI and EcoRI, PCR and sequence analysis. The recombinant plasmid containing gAd gene was transformed into E. coli BL21 (DE3), and the expression of the fusion protein His-gAd was induced by IPTG.</p><p><b>RESULTS</b>The gAd cDNA of 412 bp was obtained from the total RNA of the fat tissue and verified by sequence analysis.</p><p><b>CONCLUSION</b>The recombinant plasmid could stably express the 34-kD fusion protein His-gAd in the engineered bacteria in the form of inclusion bodies.</p>

Construction and expression of prokaryotic vector of single chain antibody derived from a new clone of anti-CD14 antibody ZCH-7-2F9 / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To construct a prokaryotic vector of ZCH-7-2F9 single chain antibody (ScFv2F9) and to obtain the ScFv2F9 protein with biological activity for further studies.</p><p><b>METHODS</b>Primers were synthesized according to the gene sequence of ScFv2F9, four tandem glycin and one serine (G4S) 3linker and multiple cloning site(MCS) of pIVEX2.3-MCS vector, which included NdeI and SmaI enzyme cleaving sites. ScFv2F9 gene was amplified through splicing by overlap extension (SOE) using the high fidelity Taq polymerase. Then the gene was cloned to pGEM-T easy and pIVEX2.3-MCS vectors. Positive recombinants (pIVEX2.3-MCS/ScFv2F9) were identified through enzyme cleaving and sequenced before expression. The recombinant plasmids were transfected into E.coli BL21star(DE3)plysS for expression. After purification with Ni+resin and renaturation in vitro, the relative molecular mass (Mr) and the binding activity of the interesting protein were determined by SDS-PAGE and flow cytometry (FCM), respectively.</p><p><b>RESULT</b>The cloned ScFv2F9 gene was identified to be functional by sequencing and expressing. The interesting protein was detected in inclusion body with a Mr of 31 000. The blocking test showed that the positive cell percentage, the mean fluorescence intensity (MFI) and the peak of channel (peak Ch) were reduced by 11.73%, 11.96% and 31.57%, respectively after once blockage by scFv2F9 protein, and 26.44 %, 21.75 % and 42.11 % after blockage twice.</p><p><b>CONCLUSION</b>The ScFv2F9 against human CD14 antigen has been successfully expressed in prokaryotic cells with partial biological activity, which lays the foundation for further studies on its immunotoxin and other kinds of engineered antibodies.</p>

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.

Prokaryotic expression of OC-IdeltaD86 (Oryzacystatin-IdeltaD86) gene and analysis of its activity / 生物工程学报

According to the amino acids sequence of OC-IdeltaD86 gene and Escherichia coli codon usage, we synthesized this gene by overlap extension PCR method with 7 oligonucleotides DNA fragments. The PCR fragment was inserted into pGEM-T-easy vector and the recombined plasmid was named pGEM-T-OC-IdeltaD86. Two oligonucleotides into which the BamH I and Xho I sites were introduced were designed and synthesized based on pGEM-T-OC-IdeltaD86 and pet21b, and the PCR fragment into which the BamH I and Xho I sites were introduced was obtained. After digesting it with BamH I and Xho I, OC-IdeltaD86 gene was cloned into the corresponding sites of pet21b and obtained prokaryotic expression vector pet21b-OC-IdeltaD86. OC-IdeltaD86 gene was expressed in E. coli (BL21(DE3)plysS) after IPTG(Isopropyl beta-D-1-thiogalactopyranoside) inducement for 5 hours. The fusion protein of OC-IdeltaD86:6His gene accounted for 11.4% of total protein and 16.4% of soluble protein, which had been successfully purified by Ni-NTA and concentrated by PEG20000. This protein can effectively inhibit papain activity in vitro and may be used in anti-nematode research in vivo.

Cloning of PD-1 gene and its prokaryotic expression in Escherichia coli / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To clone human PD-1 gene, construct a prokaryotic expression plasmid and express in E. coli.</p><p><b>METHODS</b>The human PD-1 cDNA was cloned by RT-PCR from the total RNA, which was extracted from peripheral blood lymphocyte cell of the patient with chronic hepatitis B. Recombinant PD-1 protein was been expressed and purified after the prokaryotic expression plasmid had been constructed. It was identified by SDS-PAGE, DNA sequencing and amino acid sequencing.</p><p><b>RESULTS</b>The PD-1 gene was cloned and confirmed by DNA sequencing. The recombinant protein was expressed in E. coli. The purified protein was obtained, then been confirmed by amino acid sequencing.</p><p><b>CONCLUSION</b>The human PD-1 gene was successfully cloned and expressed in E. coli, which lays the foundation for further study on the function and application of PD-1.</p>

Prokaryotic expression, purification of prM of JEV and preparation of monoclonal antibody / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To prepare monoclonal antibody (mAb) against prM epitope.</p><p><b>METHODS</b>The gene encoding prM was isolated using RT-PCR from brain of JEV infected mouse and cloned into prokaryotic expression vector pET-32a. Recombinant plasmid was transformed into E.coli BL21/DE3/LysS, then the transformed cells were expressed with the induction of IPTG. The expression and purification of the prM protein was analyzed by SDS-PAGE. The BALB/c mice were immunized with purified prM protein. Hybridoma cell lines secreting monoclonal antibodies against prM were established after cell fusion of mouse splenic cell and P3-X63-Ag8.653 cells. The specificity of mAb was identified by ELISA, Western Blot and Immunohistochemistry assay.</p><p><b>RESULTS</b>mAb against prM epitope of JEV was prepared successfully.</p><p><b>CONCLUSION</b>The obtained prM specific mAb was valuable for the prevention and dignosis of Japanese encephalitis.</p>

Prokaryotic expression and antigenic activity analysis on the matrix protein genes of two strains of human metapneumovirus recently identified in Beijing / 病毒学报

Human metapneumovirus (hMPV) is a recently identified respiratory virus more like human respiratory syncytial virus in clinical symptoms. Matrix protein (M) is one of the most important structural proteins. For further studying of hMPV, the full length of M genes from the recombinant plasmid pUCm-M1816 and pUCmM1817 were cloned by PCR and sub-cloned into the pET30a(+) vector, which is a prokaryotic expression vector, after dual-enzyme digestion with Bam HI and Xho I. The positive recombinated plasmids were transformed into E. coli BL21 (DE3) and expressed under the inducing of IPTG. Target proteins were characterized by SDS-PAGE and Western blotting. In this article, we' ve successfully constructed the recombinated plasmids pET30a-M1816 and pET30a-M1817 which have correct open reading frames confirmed by dual-enzyme digestion analysis and sequencing. The fusion proteins with 6 x His-N were highly produced after inducing by 1mmol/ L IPTG at 37 degrees C. A unique protein band with approximate 27.6 kD was characterized by SDS-PAGE. Most of the target protein existed in inclusion body. Western blot analysis showed that the target protein has specific binding reaction to rabbit antiserum against polypeptides of the matrix protein of hMPV. So the M genes were highly expressed in the prokaryotic system and the expressed M proteins have specific antigenic activities. It can be used for further studying of hMPV infections in Beijing.