Cloning And Expression Of The Extracellular Domain Fragment Of Mouse CD19 Gene And The C Terminal Domain Fragment Of Human H1e Gene

CD19 is typeⅠintegral membrane protein mainly expressed in the surface of B lymphocytes.As a member of the CD19/CD21/CD81 complex,the membrane protein complex couples the innate immune recognition of microbial antigens by the complement system to the activation of B cells.CD21 binds the C3d fragment of activated C3 that becomes covalently attached to targets of complement activation,and CD19 co-stimulates signaling through the antigen receptor,membrane immunogolbulin.The entracellular and transmembrane regions of CD19 interact with CD21 and CD81.In order to research the mechanism of CD19 in mouse immune defense,in present study,the extracellular domain gene of mouse CD19 was cloned,identified and expressed.Firstly,gene-specific primers were designed based on the putative coding region of mouse CD19 registered in GenBank and multi-clone restriction enzyme sites of prokaryotic expressive vector pGEX-4T-1.Then,total RNA was prepared from the spleen tissue of a 4-week-old mouse according to the instructions of the TRIzol reagent,the gene fragment about the extracellular domain of mouse CD19 is amplified by reverse transcription-polymerase chain reaction(RT-PCR),the result of endonuclease BglⅡdigesting RT-PCR products showed that the gene fragment has the same restriction enzyme site as known mouse CD19.The PCR products which encode the extracellular domain of mouse CD19 was cloned into pGEX-4T-1,the recombinant plasmid was identified by PCR and endonuclease EcoRⅠ+SalⅠdigestion.The result suggestes that the obtained gene fragments contain 918 base pairs and translate into protein containing 306 amino acids. The cloned mouse MECD19 shares 99.7%identity with the previously identified mouse CD19 at nucleotide level.Secondly,the recombinant,namely pGEX-4T-1-MECD19,was transformed into E.coli strain BL21 and GST-MECD19 fusion protein was induced to express.The MW of the fused protein was about 59 KD as analyzed by SDS-PAGE.And the proteins' solubility was identified,the expressed fused protein GST-MECD19 located in inclusion bodies.Finally,after optimizing prokaryotic expression conditions,we determined the optimum inducement time and concentration of isopropylthio-β-D-galactoside(IPTG).The recombinant plasmid pGEX-4T-1-MECD19 was induced to express large-scale fused protein GST-MECD19,the induced recombinant bacteria were lysed by freeze-thaw and sonication.At last,we obtained the GST-MECD 19 inclusion body protein,which could be solubilized by sonication after the detergent lauroylsarcosine was added.In conclusion,we cloned and expressed MECD19 gene in prokaryocyte successfully. All these provide some experimental materials for future studies on the immunity function of mouse CD19. Chromation consists of very long double-stranded DNA molecules and a nearly equal mass of rather small basic proteins termed histones.The histones are a group of small basic proteins(pI＞10.0)found in all eukaryotes which can tightly bound to negative charged DNA.Consisting of five major classes,the histones are amazingly similar in their primary structures among eukaryotic species.H1 proteins in eukaryotic cells may have closely related 8 subtypes whose amino acid sequences are slightly different.H1 is believed to facilitate coiling of the beaded fiber into higher-order structures.Firstly,gene-specific primers were designed based on the putative coding region of human Histl Hle registered in GenBank and multi-clone restriction enzyme sites of prokaryotic expressive vector pET-32a.Then,the human genomic DNA was prepared from the lymphocytes of normal human blood,the gene fragment about the C terminal domain of human Histl Hle is amplified by polymerase chain reaction(PCR).The PCR products which encode the C terminal domain of human Histl Hle was cloned into pET-32a,then the recombinant plasmid was identified by PCR and endonuclease EcoRⅠ+SalⅠdigestion.The result suggested that the obtained gene fragments contain 435 base pairs and translate into protein containing 145 amino acids.The cloned human Hle C shares 100% identity with the previously identified human Histl Hle at nucleotide level.The result indicates that the human Hle C gene has been successfully cloned from human genomic DNA.Secondly,the recombinant,namely pET-32a-Hle C,was transformed into E.coli strain BL21 and His-H1 e C fusion protein was induced to express.The MW of the fusion protein was about 36 000 as analyzed by SDS-PAGE.And the fusion proteins' solubility was identified,the expressed fusion protein His-Hle C located in inclusion bodies.Finally,after optimizing prokaryotic expression conditions,we determined the optimum inducement time and concentration of isopropylthio-β-D-galactoside(IPTG).The recombinant plasmid pET-32a-Hle C was induced to express large-scale fusion protein His-Hle C,the induced recombinant bacteria were lysed by freeze-thaw and sonication.At last,we obtained the His-Hle C inclusion body protein,which could be solubilized by sonication after the detergent lauroylsarcosine was added.In conclusion,we cloned and expressed Hle C gene in prokaryocyte successfully.All these provide some experimental materials for future studies on the delivery function of human Histl Hle.