Method for preparing human pregnancy-specific glycoprotein 9
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A technology of protein and recombinant bacteria, applied in the field of genetic engineering, can solve the problem of no specific antibody
Inactive Publication Date: 2014-11-19
BEIJING CHAOYANG HOSPITAL CAPITAL MEDICAL UNIV
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The mining of PSG9 function is largely limited: there is no stable and specific antibody to analyze the expressio
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Embodiment 1
[0026] Embodiment 1, the construction of human PSG9 genetic engineering strain
[0027] 1. Cloning of the full-length gene of human PSG9 by polymerase chain reaction
[0028] Using the upstream and downstream primer sequences, the full-length human PSG9 coding gene sequence was cloned using the pcDNA3.1-PSG9 plasmid vector as a template. The primer sequences are as follows:
[0029] Upstream primer sequence: 5'-CTA GCTAGC ACAGGCAGCAGAGACCATGGG-3', the underline represents the NheI restriction site;
[0030] Downstream primer sequence: 5'-CC AAGCTT TACAGTCTCAGAGTCAGTCATGA-3', the underline represents the Hind III restriction site, and the downstream stop codon was replaced with CAT.
[0031] It is also possible to artificially synthesize the gene shown in nucleotides 1-1278 in SEQ ID NO.1, and add Nhe I restriction sites and Hind III restriction sites upstream and downstream.
[0032] 2. Double digestion and ligation
[0033] The PCR amplified product was digested with ...
Embodiment 2
[0036] Embodiment 2, expression and purification of PSG9 protein
[0037] 1. Exploration of expression conditions of PSG9-6×His protein
[0038] When E.coli BL21(DE3)-PSG9-6×His was cultured until the OD value of the bacterial solution was 0.6, the target protein PSG9 was induced with the inducer Isopropylβ-D-1-Thiogalactopyranoside (IPTG) -6 x His expression. In order to obtain a high yield of PSG9-6×His protein, we optimized the concentration of IPTG and the induction temperature.
[0039] The expression of the target protein PSG9-6×His was induced by different concentrations (0 μM, 0.5 μM, 1 μM, 2 μM, 3 μM, 5 μM) of IPTG. The results showed that when the concentration of IPTG was 3 μM, the concentration of the target protein reached the maximum ( figure 2 A).
[0040] The expression of the target protein PSG9-6×His was induced by IPTG at different temperatures (20°C, 25°C, 30°C, 37°C), and the concentration of IPTG in the bacterial solution was 3 μM. The results showe...
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Abstract
The invention relates to a method for preparing human pregnancy-specific glycoprotein 9. The method comprises the following steps: (1) construction of a recombinant strain, i.e. transferring encoding gene of human PSG9 protein into a host escherichia coli to obtain the recombinant strain; (2) induction culture of the recombinant strain, i.e. conducting induction culture of the recombinant strain at the temperature of 30 DEG C by using IPTG of which the concentration in a bacterial liquid is 3mu M to obtain induced thalli; and (3) protein purification: i.e. breaking up the induced thalli to take the supernatant which is then purified by using a nickel column with elution buffer containing 40nM imidazole as the eluent, and collecting the eluent to obtain the human PSG 9 protein. Experiment results show that the human PSG 9 prepared according to the method has high purity which can reach 27.3mg/L, thereby laying a foundation for subsequent research of action mechanism of PSG 9 in cancers and obtaining of a high-purity monoclonal antibody.
Description
technical field [0001] The invention relates to the field of genetic engineering, in particular to a method for preparing human pregnancy-specific glycoprotein 9. Background technique [0002] Pregnancy specific glycoprotein (PSG) is a glycosylated protein with complex tertiary protein structure and is a member of the carcinoembryonic antigen (CEA) family. However, there are few studies on PSG family at present. PSGs contain 11 members in total, and the protein sequences among the members are highly conserved. Like the CEA family proteins, they all contain immunoglobulin-like domains (domains), which are the main structural domains of the proteins. Studies have also found that PSG1 may be involved in embryonic angiogenesis. In addition, it was found that another important function of PSG protein is that it can participate in immune response through transforming growth factor (Transfer growth factor, TGF)-beta, and can inhibit the occurrence of colonic inflammation. [000...
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IPC IPC(8): C07K14/245C12N1/21C12N15/70C12R1/19
CPCC07K14/4715
Inventor 杨磊黄骁舾王敏
Owner BEIJING CHAOYANG HOSPITAL CAPITAL MEDICAL UNIV
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