Expression of antigens of foot-and-mouth disease virus in plants: a review / 生物工程学报

Foot-and-mouth disease (FMD) is an acute, severe, and highly contagious infectious disease caused by foot-and-mouth disease virus (FMDV), which seriously endangers the development of animal husbandry. The inactivated FMD vaccine is the main product for the prevention and control of FMD, which has been successfully applied to control the pandemic and outbreak of FMD. However, the inactivated FMD vaccine also has problems, such as the instability of antigen, the risk of spread of the virus due to incomplete inactivation during vaccine production, and the high cost of production. Compared with traditional microbial and animal bioreactors, production of antigens in plants through transgenic technology has some advantages including low cost, safety, convenience, and easy storage and transportation. Moreover, since antigens produced from plants can be directly used as edible vaccines, no complex processes of protein extraction and purification are required. But, there are some problems for the production of antigens in plants, which include low expression level and poor controllability. Thus, expressing the antigens of FMDV in plants may be an alternative mean for production of FMD vaccine, which has certain advantages but still need to be continuously optimized. Here we review the main strategies for expressing active proteins in plants, as well as the research progress on the expression of FMDV antigens in plants. We also discuss the current problems and challenges encountered, with the aim to facilitate related research.

Expression of influenza B virus hemagglutinin and its immunogenicity determination / 生物工程学报

Influenza B virus is one of the causes for seasonal influenza, which can account for serious illness or even death in some cases. We tested the expression of extracellular domain of hemagglutinin (HA-ecto) of influenza B viruses in mammalian cells, and then determined the immunogenicity of HA-ecto in mice. The gene sequence encoding influenza B virus HA-ecto, foldon sequence, and HIS tag was optimized and inserted into pCAGGS vector. The opening reading frame (ORF) of neuraminidase was also cloned into pCAGGS. The pCAGGS-HA-ecto and pCAGGS-NA were co-transfected into 293T cells using linear polyethylenimine. Cell supernatant after transfection was collected after 96 h, and the secreted trimmeric HA-ecto protein was purified by nickel ion affinity chromatography and size exclusion chromatography. Subsequently, the mice were immunized with HA-ecto protein, and the corresponding antibody titers were detected by ELISA and hemagglutination inhibition (HAI) assays. The results showed that soluble trimeric HA-ecto protein could be obtained using mammalian cell expression system. Moreover, trimeric HA-ecto protein, in combination with the adjuvant, induced high levels of ELISA and HAI antibodies against homogenous and heterologous antigens in mice. Thus, the soluble HA-ecto protein expressed in mammalian cells could be used as a recombinant subunit vaccine candidate for influenza B virus.

Efficient cascade biosynthesis of (S)-2-hydroxybutyric acid / 生物工程学报

2-Hydroxybutyric acid (2-HBA) is an important intermediate for synthesizing biodegradable materials and various medicines. Chemically synthesized racemized 2-HBA requires deracemization to obtain optically pure enantiomers for industrial application. In this study, we designed a cascade biosynthesis system in Escherichia coli BL21 by coexpressing L-threonine deaminase (TD), NAD-dependent L-lactate dehydrogenase (LDH) and formate dehydrogenase (FDH) for production of optically pure (S)-2-HBA from bulk chemical L-threonine (L-Thr). To coordinate the production rate and the consumption rate of the intermediate 2-oxobutyric acid in the multi-enzyme cascade catalytic reactions, we explored promoter engineering to regulate the expression levels of TD and FDH, and developed a recombinant strain P21285FDH-T7V7827 with a tunable system to achieve a coordinated multi-enzyme expression. The recombinant strain P21285FDH-T7V7827 was able to efficiently produce (S)-2-HBA with the highest titer of 143 g/L and a molar yield of 97% achieved within 16 hours. This titer was approximately 1.83 times than that of the highest yield reported to date, showing great potential for industrial application. Our results indicated that constructing a multi-enzyme-coordinated expression system in a single cell significantly contributed to the biosynthesis of hydroxyl acids.

Gene cloning, protein expression and examination of biological activity of chicken CD40L / 生物工程学报

To obtain chicken CD40L protein, the cDNA was prepared from chicken splenic cells and used as a template to clone and amplify CD40L by PCR. The target gene was cloned into pFastBac vector to construct a pFastBac-chCD40L donor plasmid. Recombinant plasmid was transformed into DH10Bac and recombinant Bacmid-chCD40L was obtained. The Bacmid-chCD40L plasmid was transfected into sf9 insect cells to obtain His-chCD40L protein. In addition, the target gene was cloned into pQM01 vector to construct a pQM01-chCD40L plasmid, recombinant plasmid was transfected into HEK 293T cells to obtain Strep-chCD40L protein. The chCD40L protein was purified by affinity chromatography, and the concentration of purified chCD40L protein was determined to be 0.01 mg/mL. Primary cells were isolated from the bursal tissue of 3-week old SPF chickens, and the chCD40L protein was added to the culture medium to stimulate cells. The chCD40L could bind to CD40 on B cells as examined by Western blotting, indirect immunofluorescence assay and flow cytometry, suggesting that chCD40L protein is biologically active. We successfully obtained chicken CD40L protein of biological activity, which laid the foundation in the in vitro culture of primary B lymphocytes for the isolation and diagnosis of virulent IBDV.

Construction and application of recombinant human UDP-glucuronosyltransferases expression systems / 药学学报

In the research and development of new drugs, it is very important to investigate the in vitro metabolism of candidate drugs. Traditional models such as liver microsomes have many limitations, while the in vitro model of recombinant human drug metabolizing enzymes is considered as an important and useful approach because of its convenient access, stable activity and low cost. In this study, six major human UDP-glucuronosyltransferases (UGTs) genes (UGT1A1, 1A3, 1A4, 1A6, 1A9 and 2B7) were cloned from human liver cDNA and heterologously expressed in Saccharomyces cerevisiae and baculovirus-infected insect cell. UGT1A1, 1A3, 1A6 and 1A9 were successfully expressed in yeast and showed glucuronidation activity against a variety of different structural types of substrates, but their activities were low. All six UGTs were successfully expressed and exhibited significantly improved glucuronidation activity when Trichopolusia ni cells BTI-TN5B1-4 (High Five) were used as the host. The recombinant human UGTs expressed in insect cells can catalyze the glucuronidation of their specific substrates, and the glucuronidation products were synthesized at milligram-scale with yields of 13%-66% for the first time, of which the structures were identified via MS, 1H NMR, and 13C NMR spectroscopic analysis. Above all, the recombinant human UGTs yeast and insect cell expression systems constructed in this study can be used for in vitro metabolism evaluation in the early stage of new drugs research and development, and also provide a new tool for the synthesis of glucuronide metabolites.

Establishment of YAP1 knockdown system induced by tetracycline and its effect on the function of gastric cancer cells / 西安交通大学学报(医学版)

Objective: To establish a tetracycline-induced gene knockdown system and study the effect of YAP1 on the function of gastric cancer cells. Methods: We constructed pLKO.1-tetON-YAP1 knock-down lentivirus and detected the vector by enzyme digestion and sequencing. Gastric cancer cell lines SGC-7901 and MKN-28 were infected with lentivirus, and YAP1 knocked-down gastric cancer cell lines induced by DOX were established. The mRNA level of YAP1 was detected by RT-qPCR, and the protein level of YAP1 was detected by Western blotting. Cell proliferation was detected by plate cloning experiment, and cell migration was detected by scratch-healing assay and Transwell assay. Results: The results of double enzyme digestion showed two bands at 6 000 bp and 3 000 bp, and that the sequencing results were consistent with the designed shRNA sequence. In the DOX-induced group, the mRNA and protein levels of YAP1 in gastric cancer cells infected with pLKO.1-tetON-YAP1 lentivirus significantly decreased compared with those in non-induced group. In the plate cloning experiment, the number of clones in shYAP1 groups decreased significantly after DOX induction, but there was no significant change in the non-induced group. Scratch-healing assay and Transwell assay showed that after DOX induction, the cell migration ability of shYAP1 groups was inhibited, but without significant change in the non-induced group. Results: We have successfully established a tetracycline-induced lentivirus system, and knocked down YAP1 gene of gastric cancer cells with this system. The proliferation and migration of gastric cancer cells are inhibited by YAP1 in this tetracycline induced lentivirus system.

Advances in antibody drug expression techniques / 生物工程学报

The 21st century is regarded as the century of biotechnological drugs, among which monoclonal antibodies and their derived targeting drugs have established themselves as the leading modality of biopharmaceutical pharmaceutics for a wide range of indications covering malignant tumors and autoimmune disorders. Since the manufacturing of the first antibody drug from hybridoma cells, the technologies have been intensely studied and there emerged numerous breakthroughs in recombinant cell line establishment, antibody expression and purification, quality control and other related areas. This article summarizes the critical progresses of antibody drugs expression technologies, especially of mammalian cell expression system, Escherichia coli expression system, the transgenic animal reactor and the cell free protein synthesis system, to give a detailed illustration of the recent advances in antibody drugs development.

Expression, purification and characterization of recombinant PLCζ protein in baculovirus-insect cell expression system / 生物工程学报

PLCζ is a new isoenzyme of the PLC family which plays an important role in activating mammalian oocytes. In recent years, large-scale expression and purification of active PLCζ protein in vitro for structural biology research has not been successful. In this study, the recombinant human PLCζ protein was expressed and purified in the baculovirus expression system. First, the full length of human PLCζ gene was cloned into the pFastBac-HTA plasmid to form the recombinant donor plasmid that was further transformed into DH10Bac Escherichia coli cells to construct the recombined bacmid by the site-specific transposition that was screened by resistance and blue-white spots. Then the bacmid was transfected to Sf9 insect cells via cellfectin to package the recombinant baculovirus. After the amplification of the recombinant baculovirous, the recombinant protein was expressed from the cells transduced by the recombinant baculovirus and was purified by Ni-NTA resin. Purified protein was identified by Western blotting and time-of-flight mass spectrometry and the enzyme activity was determined. The results showed that the recombinant PLCζ protein in the Sf9 cells was achieved at 72 hours after baculovirus infection and expressed in secreted form in cell culture medium. The recombinant protein purified by Ni²⁺ affinity column was identified as PLCζ by Western blotting and ionization time-of-flight mass spectrometry and the enzyme activity was up to 326.8 U/mL. The experimental results provide a reference for the large-scale production and biological application of recombinant human PLCζ protein.

Heterologous expression and enhanced production of ß-1, 4-glucanase of Bacillus halodurans C-125 in Escherichia coli

Background: Recombinant DNA technology enables us to produce proteins with desired properties and insubstantial amount for industrial applications. Endo-1, 4-ß-glucanases (Egl) is one of the major enzyme involved in degradation of cellulose, an important component of plant cell wall. The present study was aimed at enhancing the production of endo-1, 4-ß-glucanases (Egl) of Bacillus halodurans in Escherichia coli. Results: A putative Egl gene of Bacillus Halodurans was expressed in E. coli by cloning in pET 22b (+). On induction with isopropyl-b-D-1-thiogalactopyranoside, the enzyme expression reached upto ~20% of the cell protein producing 29.2 mg/liter culture. An increase in cell density to 12 in auto-inducing LB medium (absorbance at 600 nm) enhanced ß-glucanase production up to 5.4 fold. The molecular mass of the enzyme was determined to be 39 KDa, which is nearly the same as the calculated value. Protein sequence was analyzed by CDD, Pfam, I TASSER, COACH, PROCHECK Servers and putative amino acids involved in the formation of catalytic, substrate and metal binding domains were identified. Phylogenetic analysis of the ß-glucanases of B. halodurans was performed and position of Egl among other members of the genus Bacillus producing endo-glucanases was determined. Temperature and pH optima of the enzyme were found to be 60°C and 8.0, respectively, under the assay conditions. Conclusion: Production of endo-1, 4 ß-glucanase enzymes from B. halodurans increased several folds when cloned in pET vector and expressed in E. coli. To our knowledge, this is the first report of high-level expression and characterization of an endo-1, 4 ß-glucanases from B. halodurans.

Study on expression and activity of combinant antimicrobial peptide in Pichia pastoris / 国际检验医学杂志

Objective To construct cecropin A-thanatin combinant gene engineering antimicrobial peptide gene CA(1-7)-T(4-19) for expression in Pichia pastoris.Methods The combinant antimicrobial peptide gene was artificially synthesized via gene splicing by overlap extension (SOE).The gene was cloned into the pPICZαA vector and transformed into Pichia pastoris X-33 by electroporation.The positive clones obtained by the screening of bleomycin resistance were induced by methanol ,and the antibacterial activity of the products was detected and the antimicrobial spectrum was established.Results The combinant peptide gene CA (1-7)-T (4-19) was successfully cloned on the carrier pPICZαA.The identification results were consistent with the pre-designed gene sequence.The combinant peptide gene was expressed under the induction of methanol ,and the minimum inhibitory concentration of 76 strains of Gram-egative and Gram-positive pathogenic bacteria isolated from the clinic was obtained ,and the minimum inhibitory concentration was up to 5 μg/mL.Conclusion A combinant genetic engineering antimicrobial peptide with antibacterial activity was obtained successfully and it had obvious inhibition effect on clinical common multidrug-resistant strains.

Expression and purification of autotoxin in sf9 insect cells / 中国生化药物杂志

Objective Production of autotoxin protein in sf9 insect cells with biological activity. Methods Autotaxin cDNA was cloned into pFastBacTMHTA from melanoma cell by extraction of total RNA using TRIzol method and RT-PCR. Bacmid-ATX is isolated from transformed competent bacterial DH10 which carries Bac genomic sequences and transfected into sf9 using lipofectamine 2000. Recombinant ATX virus was amplified in sf9 and further used for infection and expression of ATX protein. Two step purification product using HistrapTMHP and Hiload 16/600 Suerdex 200pg was determined for lysophospholipase D (lysoPLD) activity. Results Correct insertion of PCR fragment is confirmed by BamH I/Xho I digestion and sequencing. ATX virus can infect sf9 and induced enzymatic activity. Column purification and SDS-PAGE resulted 95% in purity and 6mg/liter in yield with significant lysoPLD activity. Conclusion ATX Baculovirus was successfully constructed that can infect sf9 cells and express active lysoPLD. Production of active ATX can be used for crystalography studies and screening for small pharmaceutical inhibitors.

Preparation of a polyclonal antibody against human LYZL4 and its expression in the testis / 中华男科学杂志

Objective@#To prepare a polyclonal antibody against human lysozyme-like protein 4 (LYZL4) expressed in the prokaryotic system and identify the distribution of LYZL4 in the testis.@*METHODS@#The full-length cDNA of LYZL4 was cloned into the pET32a plasmid and the expression of the recombinant LYZL4 (rLYZL4) was induced by IPTG. The rLYZL4 was purified by Ni-NTA and chitin affinity chromatography respectively and its bactericidal activity was observed by bilayer agar plate diffusion assay. The purified rLYZL4 was used as an immunogen to generate the polyclonal antibody, followed by examination of the antibody titer by ELISA and its specificity by Western blot. The distribution of LYZL4 in human tissue, sperm and seminal plasma was identified and its subcellular localization in the testis was determined by immunohistochemistry.@*RESULTS@#rLYZL4 was expressed efficiently in the prokaryotic system and exhibited no bacteriolytic activity against M. lysodeikticus and E. coli. The anti-rLYZL4 polyclonal antibody could bind the recombinant protein with a high sensitivity and specificity. LYZL4 was identified in the testis, epididymis and sperm protein extracts and localized in the acrosomal region of round and elongating spermatids.@*CONCLUSIONS@#An anti-rLYZL4 polyclonal antibody was successfully prepared using the prokaryotic expression system. LYZL4 was detected in the acrosomal region of round and elongating spermatids, suggesting an association with the structure and function of the acrosome.

Expression, purification, characterization and immunogenicity of human immunodeficiency virus-1 glycoprotein gp120 derived from insect cells / 中华微生物学和免疫学杂志

Objective To establish an efficient baculovirus-insect cell expression system for the production of human immunodeficiency virus-1 ( HIV-1 ) envelope glycoprotein gp120 and to evaluate the physiochemical properties, antigenicity and immunogenicity of the recombinant protein. Methods The gene encoding HIV-1 NL4-3 gp120 was cloned into the downstream of pH promoter of the baculovirus transfer vec-tor pAcgp67B to construct the recombinant transfer vector pAc-gp120. Expression of the protein of interest was induced in baculovirus-infected High FiveTM insect cells. ELISA, analytical ultracentrifugation and size-exclusion chromatography were carried out to characterize physicochemical properties of the expressed gp120 protein. Immunogenicity of the recombinant gp120 protein was analyzed by HIV neutralization assay after im-munizing BALB/c mice with it. Results The recombinant HIV-1 gp120 protein was successfully obtained from the established insect cell expression system with a purity of more than 90％ and a mean yield of 13 mg/L in four batches. That recombinant HIV-1 gp120 protein was characterized by homogeneity in solution and possessed a good immunoreactivity to neutralizing antibodies and antisera against HIV. Immunogenicity analysis in BALB/c mice demonstrated that the recombinant gp120 protein could induce effective immune re-sponses against HIV-1 NL4-3. Conclusion A simple and scalable approach to obtain homogeneous and im-munogenic HIV-1 gp120 antigen is successfully established, which will promote further investigation of HIV vaccine candidates.

Expression, purification, characterization and immunogenicity of human immunodeficiency virus-1 glycoprotein gp120 derived from insect cells / 中华微生物学和免疫学杂志

Objective To establish an efficient baculovirus-insect cell expression system for the production of human immunodeficiency virus-1 ( HIV-1 ) envelope glycoprotein gp120 and to evaluate the physiochemical properties, antigenicity and immunogenicity of the recombinant protein. Methods The gene encoding HIV-1 NL4-3 gp120 was cloned into the downstream of pH promoter of the baculovirus transfer vec-tor pAcgp67B to construct the recombinant transfer vector pAc-gp120. Expression of the protein of interest was induced in baculovirus-infected High FiveTM insect cells. ELISA, analytical ultracentrifugation and size-exclusion chromatography were carried out to characterize physicochemical properties of the expressed gp120 protein. Immunogenicity of the recombinant gp120 protein was analyzed by HIV neutralization assay after im-munizing BALB/c mice with it. Results The recombinant HIV-1 gp120 protein was successfully obtained from the established insect cell expression system with a purity of more than 90％ and a mean yield of 13 mg/L in four batches. That recombinant HIV-1 gp120 protein was characterized by homogeneity in solution and possessed a good immunoreactivity to neutralizing antibodies and antisera against HIV. Immunogenicity analysis in BALB/c mice demonstrated that the recombinant gp120 protein could induce effective immune re-sponses against HIV-1 NL4-3. Conclusion A simple and scalable approach to obtain homogeneous and im-munogenic HIV-1 gp120 antigen is successfully established, which will promote further investigation of HIV vaccine candidates.

Recombinant expression of hantaan virus protein N with application of Western-blot in detecting anti-hantavirus antibody / 中华流行病学杂志

Objective S gene of hantavirus(HV) was expressed in insect cells by genetic engineering technology.The expression product of S gene was used as antigen to detect anti-HV specific antibody IgG in serum.Methods Gene encoding NP of the strain HV-Z10 was amplified by PCR and then its eukaryotic expression system rBAC-Z 10S-TN was constructed by using the routine genetic engineering method.SDS-PAGE was applied to measure the expression of rNP.Ion-exchange plus Ni-NTA-affinity chromatography was performed to purify the recombinant product.Indirect immuno-fluorescence assay (IFA) was used to determine the specific immune-reactivity of rNP.WB assay was established to detect the serum samples from 95 confirmed HFRS patients.Parameters related to the outcomes of detection were compared with the routine HV-IgG IFA method.Results rBAC-Z10S-TN was able to express rNP with high efficiency.The purified rNP only showed a single protein fragment in the gel after SDS-PAGE.HV IgG could efficiently recognize rNP and hybridize with the recombinant protein.97.67％ of the serum samples from the HFRS patients were positive confirmed by WB.Conclusions We successfully constructed a high efficient prokaryotic expression system of NP encoding gene from hantavirus strain HV-Z10.WB assay which was established in this study could be used as a new serological test for HFRS diagnosis,thanks to the simplicity,safety,sensitivity and specificity of this method.

Recombinant expression of hantaan virus protein N with application of Western-blot in detecting anti-hantavirus antibody / 中华流行病学杂志

Objective S gene of hantavirus(HV) was expressed in insect cells by genetic engineering technology.The expression product of S gene was used as antigen to detect anti-HV specific antibody IgG in serum.Methods Gene encoding NP of the strain HV-Z10 was amplified by PCR and then its eukaryotic expression system rBAC-Z 10S-TN was constructed by using the routine genetic engineering method.SDS-PAGE was applied to measure the expression of rNP.Ion-exchange plus Ni-NTA-affinity chromatography was performed to purify the recombinant product.Indirect immuno-fluorescence assay (IFA) was used to determine the specific immune-reactivity of rNP.WB assay was established to detect the serum samples from 95 confirmed HFRS patients.Parameters related to the outcomes of detection were compared with the routine HV-IgG IFA method.Results rBAC-Z10S-TN was able to express rNP with high efficiency.The purified rNP only showed a single protein fragment in the gel after SDS-PAGE.HV IgG could efficiently recognize rNP and hybridize with the recombinant protein.97.67％ of the serum samples from the HFRS patients were positive confirmed by WB.Conclusions We successfully constructed a high efficient prokaryotic expression system of NP encoding gene from hantavirus strain HV-Z10.WB assay which was established in this study could be used as a new serological test for HFRS diagnosis,thanks to the simplicity,safety,sensitivity and specificity of this method.

Characterization of N-glycosylation in an anti-EGFR monoclonal antibody produced by different expression systems / 生物工程学报

The use of mammalian expression systems results in a remarkable heterogeneity of mAb products, generally due to post-translational modifications, and glycosylation is a critical post-translation modification because it has a profound impact on the safety and efficacy of mAbs. The present study was designed to explore the impact of a different expression system on mAb N-glycosylation. The detailed structures of individual glycans between anti-EGFR monoclonal antibodies produced by different expression systems were successfully characterized at the level of free oligosaccharides using liquid chromatography electrospray ionization quadrupole time-of-fight mass spectrometry (LC-ESI-QTof MS). An alternating low and elevated collision energy scan, in source collision-induced dissociation and MS/MS in combination with exoglycosidase digestion method was also adopted. The combined data revealed that the Fab region of anti-EGFR antibody produced by CHO cell expression system had a pattern of glycosylation differing from that of the SP2/0 cell expression system whereas the Fc region remained basically unchanged. We confirmed that anti-EGFR antibody produced by SP2/0 cell expression system had a much more diverse mixture of glycans with α-Gal and an undesired, aberrant form of sialylation N-glycolylneuraminic acid (NGNA). The α-Gal was absent in mAb produced by CHO cell expression system containing sialic acid predominantly N-acetyl neuraminic acid (NANA) which is the desired, normal human-type sialylation. This study theoretically predicts that anti-EGFR antibody produced by CHO cell expression system may show better clinical tolerance, and very low potential for active hypersensitivity reactions, CHO cell lines can be the preferred expression system for producing anti-EGFR biobetter.

Production of Recombinant Anti-Cancer Vaccines in Plants

Plant expression systems have been developed to produce anti-cancer vaccines. Plants have several advantages as bioreactors for the production of subunit vaccines: they are considered safe, and may be used to produce recombinant proteins at low production cost. However, several technical issues hinder large-scale production of anti-cancer vaccines in plants. The present review covers design strategies to enhance the immunogenicity and therapeutic potency of anti-cancer vaccines, methods to increase vaccine-expressing plant biomass, and challenges facing the production of anti-cancer vaccines in plants. Specifically, the issues such as low expression levels and plant-specific glycosylation are described, along with their potential solutions.

Production of Recombinant Anti-Cancer Vaccines in Plants

Plant expression systems have been developed to produce anti-cancer vaccines. Plants have several advantages as bioreactors for the production of subunit vaccines: they are considered safe, and may be used to produce recombinant proteins at low production cost. However, several technical issues hinder large-scale production of anti-cancer vaccines in plants. The present review covers design strategies to enhance the immunogenicity and therapeutic potency of anti-cancer vaccines, methods to increase vaccine-expressing plant biomass, and challenges facing the production of anti-cancer vaccines in plants. Specifically, the issues such as low expression levels and plant-specific glycosylation are described, along with their potential solutions.

Prokaryotic expression and purification of the cDNA of recombinant human cytokeratin 9 / 西安交通大学学报(医学版)

Objective To clone and fuse the cDNA of human cytokeratin 9 in prokaryotic expression system,and purify and identify the fusion protein.Methods The cDNA fragment of human cytokeratin 9 was amplified from human keratinocyte (HaCaT) total RNA with specific primers.The PCR products were cloned into vector pET-28a,then the fusion protein of his-CK9 was induced by IPTG.The expressed fusion protein of his-CK9 was purified by nickel ion affinity chromatography and identified by SDS-PAGE and Western blot.Results The sequencing proved that the recombinant vector of the cDNA of CK9 was correct.The fusion protein of his-CK9 was induced to be expressed in E.coli.The fusion protein of his-CK9 was highly purified and his-CK9 showed specific binding to the commercialized antibodies of CK9.Conclusion The recombinant vector of pET-28a-CK9 has been successfully constructed,and the fusion protein of his-CK9 has been successfully expressed and purified.