Cloning, Expression Of Sodium Pump α2 Subunit M4-M5 Intracellular Domain & PreParation And Characterization Of Monoclonal Antibody Agaist M4-M5 Recombinant Protein

Sodium pump (Na~+, K~+-ATPase), which is a special membrane binding protein that is widespread on higher eukaryotes membrane, plays an important role in the maintenance of osmotic pressure balance, membrane potential stabilization, as well as signal transduction. Na~+, K~+-ATPase molecule is a heterodimer composed ofα-subunit andβ-subunit.α-subunit is the catalytic subunit that has the enzyme activity and many binding sites of sodium pump regulation factors. It is embedded across the membrane with 10 transmembrane domains (M1-M10), of which the M4-M5 domain is directly involved in the ATP catalytic hydrolysis. There are four isoforms of theα-subunit (α1,α2,α3 andα4) found in different tissues with different function and Species specificity. Recent studies indicate that sodium pumpα2 subunit is greatly related to the occurrence and development of hypertension. In this study, the prokaryotic expression system of sodium pumpα2 subunit M4-M5 intramembrane domain protein BB4-5 was constructed and the target protein was expressed and purified, and the monoclonal antibodies agaist M4-M5 recombinant protein of sodium pumpα2 subunit were prepared, which lay the foundation for the further study of the interaction mechanism of sodium pump in the body and the relationship with the disease.1. Cloning and expression of the sodium pumpα2 subunit M4-M5 proteinObjective: To build sodium pumpα2 subunit M4-M5 intramembrane domain protein expression vector by gene recombination technology, express the protein by IPTG inducing and purify the protein by affinity chromatography, which could be used as a source of antigen for the preparation of mAb to sodium pumpα2 subunit. Methods: (1) Construct sodium pumpα2 subunit M4-M5 cloning vector. PCR was carried out with the plasmid YhNα2 (containing human sodium pumpα2 subunit cDNA sequence) as template and the gene fragments of theα2 subunit M4-M5 intramembrane domain was obtained: BB4-5. After tailing, the gene fragment are connected to pGM-T cloning vector and transformed into E.coli DH5α. The positive clones were selected and the plasmids pGMT-BB4-5 was obtained. (2) Build the sodium pumpα2 subunit M4-M5 protein expression system. The target gene fragments was cut from T vector, and connected into the expression vector pET28b (+) by double digestion and transformed into E. coli Rosetta2. The positive clones were selected and the expression plasmids pET28b (+)-BB4-5 was obtained. After verifying the sequence of the target gene by double digestion, the expression bacteria were inoculated and induced by IPTG with a final concentration of 0.4mM/L and SDS-PAGE was carried out to detect the expression of recombination protein. (3) The purification of recombinant protein: E.coli was broken and the exist form of tagart protein was identified by SDS-PAGE. After denaturation and renaturation of the inclusion bodies, the target protein was purified by Ni-NTA affinity chromatography and the purity was tested. (4) Western blotting analysis of recombinant protein: After SDS- PAGE, the recombinant protein purified by Ni-NTA affinity chromatography was electrotransferred to nitrocellulose membrane, blocked and incubated with sodium pumpα2 antiserum or anti-His antibody. Then the membrane was washed, the fusion protein was stained by DAB kit, and the strips were scanned by Gel Imaging analysis System.Results: (1) PCR was carried out with the plasmid YhNα2 as template and gene fragments BB4-5 was obtained successfully. PCR productions were identified by 1% agarose gel electrophoresis with clear strap in the right position. After tailing, the PCR productions are connected to pGM-T vector and transformed into E.coli DH5α. The positive clones were amplified and plasmids were extracted. Restriction enzyme digestion and sequencing results showed that plasmid sequences are consistent with the theoretical sequences. (2)BB4-5 fragment were linked to PET28b(+) expression vector which was double-digested with the same enzyme and transformed into Rosetta2 competent cells. Recombinants were screened on the plate containing Kan. Double-digestion results showed that the protein expression system was constructed successfully. After IPTG induction, SDS-PAGE analysis showed that the molecule weight of BB4-5 fusion protein is the same as expected. (3) SDS-PAGE analysis showed that expression products were mainly in the form of inclusion bodies after broken. The inclusion bodies were denaturated with guanidine hydrochloride and renaturated by gradient dilution, and the expressed products were purified by Ni2+-NTA affinity column with a purity of above 90%. (4) Western blotting analysis showed that BB4-5 recombination protein could specifically bind to sodium pumpα2 antiserum and anti-His antibody.Conclusion: The expression vector pET28b(+)-BB4-5 of the sodium pumpα2 subunit M4-M5 intramembrane domain was constructed successfully. After IPTG induction and purification by Ni-NTA affinity chromatography, the target protein BB4-5 was obtained, which could be used as a source of antigen for the preparation of mAb to sodium pumpα2 subunit.2. Preparation and characterization of monoclona antibody agaist sodium pumpα2 subunit M4-M5 recombinant proteinObjective: To use sodium pumpα2 subunit M4-M5 recombinant protein BB4-5 as an antigen to immunize BALB/c mice and prepare monoclonal antibody through hybridoma technique, and to identify the specificity of the McAb by ELISA and Western blotting, which lay the foundation for the study of mechanism of action of the sodium pump in the body and the relationship with the disease.Methods:(1) The 6~8 weeks BALB/c mice were immunized with recombinant protein BB4-5 of sodium pumpα2 subunit M4-M5 intramembrane domain as an antigen. (2)Screen the McAbs agaist sodium pumpα2 subunit with hybridoma technique: The splenocytes of the immuned mouse were separated, and then were fused with Sp2/0 myeloma cells. The positive holes were screened by indirect enzyme linked immunosorbent assay (ELISA) and the positive hybridoma clones that can stably secret McAbs were obtained through cloning by limiting dilution. (3) The monoclonal antibodies were prepared in mice injected intraperitoneally with hybridoma cells and McAbs were purificated by caprylic acid ammonium sulfate fractionation. (4) The subtypes of the McAbs were identified by Mouse Monoclonal Antibody Isotyping Kit. (5)The detection of the monoclonal antibodies specificity was performed with western blotting. Western blotting analysis was performed to detect the specificity of the McAbs. (6)Accroding to Beatty's method, the affinity constants of the McAbs were measured with indirect enzyme-linked immunosorbent assay.Results:(1) The 6~8 weeks BALB/c mice were immunized and the titer of the antiserum was above 1:50000. (2) Mice with higer antibody titer were selected and the splenic cells were separated, and then the splenocytes were fused with Sp2/0 myeloma cells by hybridoma technique. 14 positive wells were screened by indirect enzyme linked immunosorbent assay (ELISA) and 2 hybridoma cell lines were selected with high titer after the cloning by limited dilution method. (3) BALB/c mice were injected intraperitoneally with hybridoma cells and McAbs were purificated by caprylic acid ammonium sulfate fractionation. (4)The subtypes of two strains of McAbs were IgG2a that identified by Mouse Monoclonal Antibody Isotyping Kit. (5) Western blotting results indicated that all of the two McAbs strongly and specifically reacted with sodium pumpα2 subunit M4-M5 intramembrane domain. (6)The affinity constants of the two McAbs were 3.51×108L/ mol and 5.88×107 L/ mol respectively.Conclusion: Two strains of McAbs were obtained successfully by hybridoma technique with sodium pumpα2 subunit recombinant M4-M5 protein BB4-5 as an antigen, and the specificity of the McAb were identified by indirect ELISA and western blotting, which lay the foundation for the study of mechanism of action of the sodium pump in the body and the relationship with the disease.