Preparation And Application Of Antibody Against Human Zinc-Fingers And Homeoboxes

Zinc-fingers and homeoboxes 2(ZHX2) is a transcriptional repressor,which was cloned in 2003.AFP,GPC3 and H19,known as targets of ZHX2,were highly expressed in HCC.Our lab have provided direct evidence that ZHX2 repressed AFP in human liver tumors,and suppressed hepatoma cells growth.Clinical study showed that ZHX2 expression was negatively correlated to the risk of multiple myeloma. Therefore,ZHX2 might be used as a diagnostic marker and an antibody against ZHX2 with good specificity will be very helpful for the diagnosis of tumors.AimTo prepare ZHX2 polyclonal antibody and detect ZHX2 expression in cell cultures and tumor samples.Methods1 Expression of GST-ZHX2 fusion proteinThe plasmids,pGEX-ZHX2(1-837AA),pGEX-ZHX2(263AA-497AA) and pGEX-5X-1 were transformed into E.Coli BL21(DE3).The positive clones were identified by restrictive enzymes assays and automatic DNA sequencing respectively. Expression of GST and recombinant protein fused with GST tag,GST-ZHX2 (1-837AA),GST-ZHX2(263-497AA),were induced by IPTG(1mM) for about 6 hours.The cell lysate were loaded on 12%SDS-PAGE gels,followed by coomassie brilliant blue staining.Protein expression level was analyzed by Quantity One.2 Immunization of rabbits and immunoglobulin purificationThe target bands(GST,26KDa;GST-ZHX2(1-837AA),118KDa;GST-ZHX2(263-497AA),52KDa) were cut off from stained gels and triturated for more than 10 min, then stored at -20℃. The triturated suspensions of target antigen in 0.9%NaCl was used for immunization.The homogenate was used to immunize male New Zealand white rabbits.Four rabbits were inoculated at multiple sites in the back,with the target protein(200μg/rabbit) mixed with equal volume of Complete Freund's Adjuvant.And the booster immunizations with equal volume of the expressed protein mixed with Incomplete Freund's Adjuvant were carried out every two weeks for three times.Two weeks after the final boost,the serum was separated from blood by storing the blood.The serum immunoglobulin obtained was skimpily purified by saturated ammonium sulfate(SAS) precipitation method(purified three times using 50%,33% and 33%(v/v) of SAS,respectively),and sodium azide(NaN3) was added as a preservative at a final concentration of 0.02%(w/w).The purified serum was then subpackaged and stored at -80℃.3 Specificity and Sensitivity assay for ZHX2 polyclonal antibodyIn order to assay the specifiity and sensitivity of self-made antibody,HepG cells was transiently transfected with 1.0μg of pcDNA3-ZHX2-HA or pcDNA3 using Lipofectamine TM2000.Forty-eight hours after transfection,the cells were collected to detect the expression of the protein ZHX2-HA by ZHX2 pAb and HA mAb.4 Application of ZHX2-pAb(1) ZHX2 expression in different tumor cell lines.Human hepatoma cell line HepG2(ATCC HB-8065),HepG2.215 and Bel-7402, epithelial human Caucasian lung carcinoma cell line(A549),cervical cancer cell line(Hela),human gastric cancer cell lines(MGC-803 and SGC-7901),human adenocarcinoma cell line(SKOV-3) were cultured and collected for western blot. Endogenous ZHX2 protein was probed using ZHX2 pAb.(2) ZHX2 expression in HCC samples.Liver samples from 10 patients with HCC,who underwent hepatectomy in the Shandong provincial hospital,Jinan,China were obtained.Clinical data backuped from medical records.These fresh samples were washed by Rnase free PBS in order to remove the humor fluid as much as possible and then frozen immediately in liquid nitrogen and stored at -80℃for use.Protein were extrzctd from all HCC samples for western blot.Endogenous ZHX2 protein was probed using ZHX2 pAb.Results1 Expression of GST-ZHX2 fusion proteinRestrictive enzymes assay and automatic DNA sequencing confirmed the successful construction of plasmid pGEX-ZHX2(1-837AA) and pGEX-ZHX2 (263-497AA).12%SDS-PAGE showed the specific band of GST-ZHX2(1-837AA) at 118KDa and GST-ZHX2(263-497AA) at 52KDa.Protein expression level was analyzed by Quantity One.The expression of the recombinant GST-ZHX2(263-497aa) was estimated up to 10.86%of the total expressed bacterial protein,while the expression of GST-ZHX2(1-837AA)is about 5.4%.These data would be used in evaluating dosage of antigen for immunization.2 Purification and characterization of ZHX2 pAbSerum against human ZHX2,named ZHX2-pAb,was concentrated and simply purified by SAS precipitation method.To further study the specificity and sensitivity of our self-made ZHX2-pAb, transfected pcDNA3-ZHX2-HA was transfected into HepG2 cells which have less endogenous expression of ZHX2.Western blot results showed that the recombinant ZHX2-HA protein in HepG2 transfected with pcDNA3-ZHX2-HA could be detected by both ZHX2-pAb and anti-HA,but not showed in both pcDNA3-transfected HepG2 and non-transfected HepG2.All these evidence proved that our self-made ZHX2-pAb had good specificity and sensitivity for human ZHX2 detection.3 Application of ZHX2-pAb(1) ZHX2 expression in different tumour cell linesNine tumour cell lines were detected for ZHX2 protein expression.Very interestingly,ZHX2 could be detected using self-made ZHX2-pAb in all cell lines, including liver cancer,lung cancer,cervical cancer and gastric cancer cell lines. ZHX2 expression varied greatly in four HCC cell lines which is consistent with our previous results.These indicated the potential use of our self-made ZHX2-pAb in further study for ZHX2. (2) Detection of ZHX2 in HCC samplesZHX2 protein expression were also detected in 10 HCC samples,which obtained from Shandong provincial hospital,Jinan,China.The results showed that 2 samples have high expression levels of ZHX2,5 samples have a middle level,while the others have much lower levels.This indicated that our self-made pAb can be used in clinical detection.ConclusionWe succesufuly prepared polyclonal antibody against human ZHX2 and developed a approach for preparing polyclonal antibody:cutting off fusion proteins from SDS-PAGE gel,injecting protein-mixed gel into rabbits,and then concentrating antibody by SAS,which can be accomplished in all laboratory.Western blot results of transfected HepG2 cells show the good quality of the antibody.We established the western blot assay with our self-made pAb to detect ZHX2 expression in both tumor cell lines and clinical tissues.Further study will focus on the correlation ship between ZHX2 expression and disease prognosis.