Construction of a New T-Vector: Nickase (Nt.BspQI)-Generated T-Vector Bearing a Reddish-Orange Indicator Gene

T-vectors are widely used for cloning the polymerase chain reaction (PCR) products. However, the low conversion efficiency of a plasmid into the linear T-vector usually results in non-recombinants. Here, we designed a new plasmid pNBQ-T to easily select the recombinant colonies harboring PCR products. pNBQ-T plasmid, which contains a DsRed indicator gene between two Nt.BspQI restriction cassettes, each of which contains palindromic sequences susceptible to Nt.BspQI nickase (5′-GCTCTTCT

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.

A Secretive Pichia pastoris Expression Vector for Direct PCR Product Cloning / 中国生物工程杂志

A secretive expression vector of Pichia pastoris system which can be used for the direct cloning of PCR products was constructed,and was verified through the expression of recombinant cellobiohydrolase II in Pichia pastoris.A randomly selected fragment was amplified with properly designed primers by PCR.The XhoI and Eam1105Ⅰ restriction sites were included in the 5'end of the fragment,and the Eam1105Ⅰ and XbaI restriction sites were included in its 3'end.The PCR amplified product was inserted into the P.pastoris expression plasmid pPICZ?A through XhoI and XbaI restriction sites and the resultant plasmid was digested with Eam1105Ⅰ,and lastly the big fragment was recovered,generating the P.pastoris expressive Tvector pPICZ?T.Then the cellobiohydrolase II of T.reesei was successfully expressed in P.pastoris with this expressive Tvector.Such results indicated that the constructed expression Tvector was convenient for PCR product cloning,and was effective for heterologous protein expression in P.pastoris.On the other hand,the application of the expression Tvector avoided the introduction of additional amino acids at the Nterminus of the expressed protein,which generally occurred when normal expression vectors were used in secretive expression system.

Amplification of All Immunoglobulin V Genes of Human Atherosclerosis by Polymerase Chain Reaction / 上海第二医科大学学报

Objective To amplify all immunoglobulin V genes of human atherosclerosis by PCR and to sequence the products of PCR cloned into T-vector. Methods Peripheral blood samples of 50 patients with atherosclerosis were collected,from which lymphocytes were segregated by density gradient centrifugation and total RNA was extracted by TRIzol reagent.V genes of VL(including VK and V?) and VH were amplified by RT-PCR,and the products were digested by restriction enzyme and then cloned into T-vector.Two clones were picked randomly to sequence.(Results)Total RNA were extracted purely with integrity,and all V genes of VL and VH were amplified by PCR successfully.The sequences were highly homologous to human immunoglobulin genes. Conclusion All immunoglobulin V genes of human artherosclerosis were amplified successfully,which lays a foundation for the construction and (selection) of phage library.

Cloning and sequencing of the 16s rRNA intergenic spacer region of proteus mirabilis and acinetobacter iwoffii / 重庆医科大学学报

Objective:To clone and sequence of 16S~23S r RNA intergenic spacer region of Proteus mirabilis and Acinetobacter lowffii for further identification of these two bacteria by means of molecular probe.Methods: The PCR universal clinical common pathogenic bacteria primers for 16S and 23S rRNA's conserved sequences were designed.Then the genomes of these two bacteria were amplified,T Vector clones were constructed with PMD-18 T Vector ad PCR products.The vectors were sequenced and the sequence was submitted to Genbank.Results: T Vector clones were constructed with products amplified by using universal primers;according to Blast analyses,the results of sequencing were determined as ISR sequences of the two kinds of bacteria.And the reports to Genbank were accepted.Conclusion: Genbank accepted the cloning and sequencing results of Proteus mirabilis and Acinetobacter lwoffii as new sequences.And these sequences can be used to identify the two bacteria by universal primers.

THE CONSTRUCTION OF A NEW TYPE OF T-VECTOR / 微生物学通报

The template-independent teminal transferase activity of Taq DNA polymerase results in an overhanging dA at the 3′end of its PCR products. The pGEMX vector constructed in this experiment forms a single overhanging dT at its 3′end as the result of cleavage with Xcm I restriction enzyme. This vector is very efficient for direct cloning of PCR product obtained by using Taq DNA polymerase.Recombinant colonies can be selected by Blue/white screening. Moreover,insertion fragment can be easily released from the vector simply with either BamH I or Hind III digestion.

A study on the preparation of STR allelic ladder / 中国法医学杂志

Objective To produce the standard allelic ladder by using the cloning technique. Methods After the amplification and separation of the STR alleles, they were purified and then connected with T-vectors directly. The combinants were transfected into the component E. coli DH5? cells follwed by cloning and plasmid purification. The allelic ladder were then produced by re-amplifying the recombinant plasmid DNA. Results The allelic ladder made in this way can be produced in a lager amount and can be stored in a relatively long period. Conclusion The results demonstrated that the standard allelic ladder generated in this way is more practical in forensic scienc application. This technique in useful for preparation of domestic STR kits.