cDNA cloning and prediction of antigenic determinants of protein marker IP4 in Ophiocordyceps sinensis / 中草药

Objective To clone the full-length of cDNA protein marker cyanase (IP4) of Ophiocordyceps sinensis and predict the antigenic sites. Methods The information of protein marker of O. sinensis was obtained by proteomics technology. The transcriptome database of O. sinensis and mycelium constructed in our lab were used to analyze IP4 unigenes and appropriate primers designed to amplify IP4, which was then cloned and sequenced. The conserved sequence of IP4, homology comparison, and the antigenic site were analyzed by DNAMAN6.0 and DNAStar software. Results Two alternative splice variants of IP4 were discovered from the transcriptome data and belonged to the invariant alternative splicing. The protein marker of O. sinensis was identified as cyanate hydratase, 465 bp, encoding 154 aa. The analysis of the conserved and functional regions showed that catalytic site and binding site mainly lied in C-terminal of IP4. Analysis of DNAMAN 6.0 showed that species 1-39 aa and 55-81 aa were of higher species specificity, and DNAStar results showed that the epitope region of IP4 protein is distributed from 25 to 90 aa. Conclusion The optimal antigen region of IP4 lied in the region 25-39 and 55-81 aa, which lays a foundation for the subsequent large-scale preparation of IP4 protein and eutilizing ELISA to identify authenticity of O. sinensis.

Study on bio-synthesis of cordycepin in Ophiocordyceps sinensis based on RNA-seq / 中草药

Objective Recent years, some studies have been studied on the biosynthesis of cordycepin, but it is not clear. To sequence the transcriptomes of the Ophiocordyceps sinensis which could provide the basis for revealing the bio-synthesis mechanism of cordycepin. Methods In this study, by Illumina/Solexa HiSeq 2500 technology, the transcriptomes of the O. sinensis fungus (anamorph) and the fruiting body (teleomorph) was sequenced, assembled and analyzed. By RT-PCR, the full lengths of RNRL (RNR large subunit) and RNRM (RNR small subunit) cDNA were cloned from the fresh O. sinensis fruit body. Results The pathway and the genes involved in cordycepin biosynthesis were predicted. Among of them, RNR was the critical enzyme in the metabolism of adenosine, also predicted to play an important role in the biosynthesis of cordycepin. From the transcriptome data, one large, one small subunits, and four similar sequences of RNR were found. RNRL mRNA was 2 733 bp, encoding 910 aa and RNRM mRNA 1 257 bp, encoding 418 aa. The analysis of the conserved and functional regions showed that catalytic site and binding site mainly lied in RNRL, RNRM contained a ferritin-like conserved sequence. Conclusion This study would be established for revealing the bio-synthesis mechanism of cordycepin.

Construction and identification of infectious full-length cDNA clone of enterovirus 71 / 中华微生物学和免疫学杂志

Objective To construct an infectious full-length cDNA clone of enterovirus 71(EV71)and develop a technological platform for study on vaccine development as well as molecular virology of EV71.Methods According to the nucleotide sequence of EV71 strain 085 isolated in China,four pairs of primers were designed for amplification of four end to end overlapping subgenomic cDNA fragments,the cDNA fragments were directional cloned into pBluescript SK(+)vector,and the virus genome cDNA clone was obtained by ligation orderly.The rescued virus of parental strain 085 from RNA transfected host cells was identified by RT-PCR,IFA,titration as well as transmission electron microscope(TEM)after the transcription of the full-length cDNA clone in vitro.Results The full-length cDNA clone was constructed successfully,and the typical CPE was observed after its transcription into Vero cells.The rescued virus with 20-30 nm in diameter can not only be neutralized by EV71 special anti-serum but also react with anti-EV71 monoclonal antibody that virus infected cells stained with FITC can be detected by IFA.After amplification from the total RNA extraction of virus infected cells by RT-PCR with EV71 special primers,the 226 bp products can be detected.The growth curve showed that the rescued virus can propagate in Vero cells stably with a titer of 4.5 ～6.0 lgCCID50/ml during 8 passages.The plaque formed by rescued virus is identical as parental virus in morphology but smaller in size.Conclusion An infectious full-length clone of EV71 was developed successfully,which will be used for further study on pathogenesis and vaccine development of EV71.

Molecular cloning,sequence analysising and repairing the mutation of mouse integrin ?_7 gene / 重庆医科大学学报

Objective:To clone and analyze a full-length cDNA encoding mouse integrin ?7,and repair the mutation of ?7 cDNA that caused the change of amino acids. Methods:The cDNA of ?7 gene was amplified by RT-PCR using the total RNA as extracted from mouse small intestine Peyer’s patch. The PCR product was inserted into pMD19-T vector and then transformed E.coli JM109. The positive recombinant clone was analyzed by restriction endonuclease and DNA sequencing. The mutation of ?7 cDNA that caused the change of amino acids was repaired. Results:The cDNA of mouse ?7 has an complete open reading frame with a length of 2418 bp,which encodes a product of 806 amino acid,and has 10 base pairs mutation of ?7 gene and 5 base pairs mutation that caused the change of amino acids was repaired. Conclusion:The cDNA of mouse ?7 was cloned successfully,which posed a basis for further researching on its biological function.

Cloning and sequencing of the full-length of human PPAR? cDNA / 重庆医科大学学报

Objective:To clone and sequence the full-length of human PPAR? cDNA in order to further construct the eukaryotic expression vector carrying hPPAR? gene.Methods:hPPAR? cDNA was cloned from HepG2 cells total RNA by RT-PCR.The PCR product recovered from gel were ligated with pMD19-T vector and transformed into DH5? competent cell.The integrity and fidelity of hPPAR? cDNA sequence inserted in T vector were verified by BamH I and Sal I double excising and DNA sequencing assays.Results:The positive clone T vector plasmid containing correct sequence of hPPAR? cDNA were verified by enzyme digestion as well as sequence analysis and was named as pMD19-hPPAR?-T vector.The sequence of inserted hPPAR? cDNA was in accordance with the corresponding sequence in GeneBank database(AY919140).Conclusion:hPPAR? gene is successfully cloned and the pMD19-hPPAR?-T intermediate vector is constructed,which provide a good basis for further constructing the eukaryotic expression vector carrying hPPAR? gene and studying the receptor's function.

Construction and Identification of Recombinant Expressing of Human Sjogren's Syndrome Antigen A / 医学研究杂志

Objective To clone human Sjogren's syndrome antigen A(SSA)for expressing of antigen SSA-52kD and establishing a new clinical detecting method.Methods According to the human SSA-52kD cDNA sequence reported in GenBank,primers of human SSA-52kD cDNA were designed and synthesized.Human SSA-52kD cDNA was amplified from RNA of cultured Hela cell by reverse transcriptase polymerase chain reaction(RT-PCR).The production of amplification was ligated to PET-30a vector and then transformed into the competent bacteria DH5?to construct the recombinant plasmid PET-30a-SSA-52kD.The recombinant plasmid was digested with Bgl Ⅱ and Hind Ⅲ,and positive clones were sequenced.Results The Human SSA-52kD cDNA fragment containing 1447bp was amplified by RT-PCR.Restriction endonuclease mapping using Bgl II and Hind III showed that the target gene was inserted into the recombinant plasmid.The complete coding sequence of Human SSA-52kD was consistent with that of GenBank through DNA sequencing.Conclusions The full length of human SSA-52kD cDNA was successfully cloned and the recombinant plasmid PET-30a-SSA-52kD was constructed.

Construction of A Stable Full-Length cDNA Clone of Japanese Encephalitis Virus Strain SA14-14-2 Using Low Copy Number Plasmid

Recently the reverse genetics system contributed to the progresses in the investigation of positive-stranded RNA viruses. Here, we report the successful construction of a stable full-length infectious cDNA clone of the live attenuated JEV vaccine strain SA14-14-2. The eleven kilobase viral RNA genome was reverse transcribed, amplified as four overlapping DNA fragments and successively ligated into the low copy number plasmid pACYC184, which contains the p15A origin of replication. In vitro-transcribed RNAs had a specific infectivity of approximately 104 PFU/microgram RNA, and the resulting virus exhibited growth kinetics and plaque morphology similar to the parental virus in cell culture. The structural and functional integrity of the cDNA clone was stably maintained even after at least 150 generations in Escherichia coli strain TOP10. The cDNA clone was engineered to contain single nucleotide change to create a XhoI site and knock out a XbaI site (A to C at nt 9134) acting as a genetic marker. This genetic marker was retained in the recovered progeny virus. Our results suggest that the instability of the full-length infectious JEV cDNA clone can be overcome by employing low copy number plasmid pACYC184. This infectious JEV cDNA clone will aid future studies of pathogenesis, virulence, and replication. Furthermore, it will facilitate the development of SA14-14-2 based recombinant vaccines.

Construction of A Stable Full-Length cDNA Clone of Japanese Encephalitis Virus Strain SA14-14-2 Using Low Copy Number Plasmid

Recently the reverse genetics system contributed to the progresses in the investigation of positive-stranded RNA viruses. Here, we report the successful construction of a stable full-length infectious cDNA clone of the live attenuated JEV vaccine strain SA14-14-2. The eleven kilobase viral RNA genome was reverse transcribed, amplified as four overlapping DNA fragments and successively ligated into the low copy number plasmid pACYC184, which contains the p15A origin of replication. In vitro-transcribed RNAs had a specific infectivity of approximately 104 PFU/microgram RNA, and the resulting virus exhibited growth kinetics and plaque morphology similar to the parental virus in cell culture. The structural and functional integrity of the cDNA clone was stably maintained even after at least 150 generations in Escherichia coli strain TOP10. The cDNA clone was engineered to contain single nucleotide change to create a XhoI site and knock out a XbaI site (A to C at nt 9134) acting as a genetic marker. This genetic marker was retained in the recovered progeny virus. Our results suggest that the instability of the full-length infectious JEV cDNA clone can be overcome by employing low copy number plasmid pACYC184. This infectious JEV cDNA clone will aid future studies of pathogenesis, virulence, and replication. Furthermore, it will facilitate the development of SA14-14-2 based recombinant vaccines.

Molecular cloning, sequence analysis of mouse integrin ?_4 gene and repairing of its mutation / 第三军医大学学报

Objective To clone and analyze the full-length cDNA of mouse integrin ?4, and repair the mutation sensible locei that caused the change of amino acids. Methods The cDNA of ?4 gene was amplified by RT-PCR using the total RNA extracted from mouse small intestine peyer’s patch. The PCR product was inserted into pMD19-T vector and then transformed into E. coli JM109. The positive recombinant clone was analyzed by restriction endonuclease and DNA sequencing. The mutation of ?4 cDNA that caused the change of amino acids was repaired. Results The cDNA of mouse ?4 had a length of 3 099 bp, and encoded a product of 1 032 amino acids. There were 12 bases pairs mutation of ?4 gene and the 6 base pairs causing the change of amino acids was repaired. Conclusion The cDNA of mouse ?4 is cloned successfully.

Modification of the Full-length cDNA Clone of Newcastle Disease Virus of Goose Origin~* / 微生物学通报

The 6.5kb specific fragment containing the T7 promoter and the transcription vector was cut down from the full-length cDNA clone of Newcastle disease virus strain ZJI of goose origin,and thereafter it was self-ligated to form the high guality plasmid for mutagenesis.Site-directed mutagenesis technique was used for inserting three additional G nucleotides(nts) into the region between the T7 promter and the leader sequence of the NDV.The RT-PCR was employed to amplify the F/HN genes fragements,and then they were ligated by the shairing restriction enzyme BsmBI and finally the corresponding fragment in the mutatant full-length cDNA was substituted by the new one.The sequencing results showed that the three additional G nts were successfully inserted and the mutant nts in the full-length cDNA were corrected and all these studies lay a foundation for the research on the reverse genetics of NDV strain ZJI.

Cloning of human REG ? cDNA and construction of the stable transfective cell line / 重庆医科大学学报

Objective:To construct the stable transfective cell line with the eukaryotic expression vector for human REG?cDNA to laid the foundation for further study of the function of REG?.Methods:REG?cDNA was obtained by RT-PCR of total RNA extracted from the human breast cancer cell MCF-7,and it was digested by the restriction endonuclease EcoRⅠand EcoRⅤbefore connection withPcDNA3.1.The recombination with forward insert were slelected by restriction endonuclease digestion and confirmed correct by sequencing and then transfected into HBL-100 cell by using lipofectamine2000.The stable transfected cell line was selected in medium containing antibiotic(G418).Results:The result of immunocytochemistry and RT-PCR of total RNAextracted from the stable transfected cell line showed that there existed the overexpression of REG?cDNA in it.Conclusion:The construction of the eukaryotic expression vector for REG?was successful,and the stable transfected cell line for overexpression REG? was obtained by G418 selection.This work may pave the way for further study of the functions of REG?.

Direct cloning of the newest angiogenesis inhibitor,arresten / 重庆医科大学学报

Objective:To clone and express the new angiogenesis inhibitor,arresten.Methods:The total RNA was extracted from the normal human liver,and then was reversely transcribed mRNA to cDNA and arresten cDNA was amplified by nestle-PCR.After being treated by T4 DNA polymerase,the arresten cDNA was linked with the linear vector pTYB1 digested by NdeI and EcoRI.And then the complete ER2566 was transfected by recombinant vector,the recombination was screened and the expression of the interest protein was induced by IPTG.Results:The arresten was cloned by one-step cloning method with PCR product treated by T4 DNA polymerase.Conclusion:This method can be used to clone the interest gene more easily and quickly than T-A clone and others.