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Recombinant plasmid containing human SMIM25 gene, genetic engineering bacteria, recombinant protein, polyclonal antibody and preparation method and application thereof

A technology of SMIM25 and genetically engineered bacteria, applied in the field of bioengineering, can solve the problems of insufficient specificity, less than 30% 5-year survival rate of gastric cancer patients, and few markers

Active Publication Date: 2019-07-12
WEIFANG MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, there are not many markers for early diagnosis of the disease, and the specificity is not enough, which leads to the fact that some patients are already in the middle and late stages of the disease when they are diagnosed with gastric cancer
In addition, due to tumor recurrence and metastasis, the 5-year survival rate of patients with advanced gastric cancer is less than 30%
[0003] The human SMIM25 gene is a hypothetical gene included in the NCBI database. Bioinformatics analysis shows that the gene encodes a transmembrane protein and belongs to the SMIM protein family, but there is no relevant polypeptide or experimental data related to its biological function.

Method used

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  • Recombinant plasmid containing human SMIM25 gene, genetic engineering bacteria, recombinant protein, polyclonal antibody and preparation method and application thereof
  • Recombinant plasmid containing human SMIM25 gene, genetic engineering bacteria, recombinant protein, polyclonal antibody and preparation method and application thereof
  • Recombinant plasmid containing human SMIM25 gene, genetic engineering bacteria, recombinant protein, polyclonal antibody and preparation method and application thereof

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preparation example Construction

[0050] The present invention also provides a method for preparing the above-mentioned recombinant plasmid, in which a human SMIM25 gene fragment is introduced into the basic plasmid to construct the recombinant plasmid.

[0051] The preparation method of the above-mentioned recombinant plasmid provided by the present invention has simple process, convenient operation, low requirements for technical personnel, strong universality, high success rate of preparing the target recombinant plasmid, and is suitable for actual production.

[0052] Preferably, the basic plasmid is pGEX-4T-1.

[0053] The expression vector contains a tac promoter, which can achieve chemically induced high-level expression; at the same time, it has a GST tag, and the fusion protein can increase the solubility of the target protein; the obtained fusion protein of the target protein is convenient for further processing under mild conditions. Column purification; using anti-GST tagged antibody can be easily ...

Embodiment 1

[0082] Example 1 Prokaryotic expression vector construction and sequencing identification

[0083] 1.1 According to the human SMIM25 cDNA sequence included in GenBank, the whole gene was synthesized, and BamHI and XhoI restriction sites were introduced before the start codon and after the stop codon, respectively.

[0084] 1.2 Construction and identification of cloning vectors

[0085] The pGEX-4T-1 and the synthetic SMIM25 whole gene fragment were double digested with BamHI and XhoI, and the target fragment was recovered. T4 DNA ligase was ligated overnight at 16°C, and the ligated product was transformed into Escherichia coli DH5H5α competent cells. Positive clones were screened with ampicillin (100 μg / ml), plasmids were extracted in small doses by alkaline lysis, and sent to Shanghai GenScript Company for sequencing identification. Sequencing results such as Figure 1A and 1B As shown in -1D, the result shows that the sequencing of the recombinant plasmid provided in thi...

Embodiment 2

[0086] Induced expression and identification of embodiment 2 recombinant protein

[0087] Transform the competent cells of the recombinant pGEX-4T-1-SMIM25 with the correct sequence expression strain BL21 (DE3) to induce expression of the fusion protein: inoculate a single colony transformed with the recombinant plasmid (pGEX-4T-1-SMIM25) in 5m1 LB (Amp+) liquid medium, 37°C, 160rpm, shaking culture overnight. The next day, replant in fresh LB (Amp+) liquid medium at a ratio of 1:100, culture at 37°C, 220rpm for 3h until the OD600 is about 0.6, add IPTG to a final concentration of 1mM, continue to culture for 5h, and induce Expression of SMIM25 recombinant fusion protein. The culture products were collected, the culture medium and the bacteria were separated, the supernatant and the precipitate were separated after the bacteria were ultrasonically disrupted, and the presence or absence of fusion expression of the target protein and the distribution of the protein were analyze...

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Abstract

The invention provides a recombinant plasmid containing a human SMIM25 gene, a genetic engineering bacterium, a recombinant protein, a polyclonal antibody, a preparation method and application thereof, and relates to the technical field of biological engineering. The recombinant plasmid contains a human SMIM25 gene fragment; and the recombinant plasmid is low in production cost, and can be inducedin escherichia coli to generate recombinant fusion protein, and used as an antigen to prepare the SMIM25 polyclonal antibody so as to rapidly and accurately detect SMIM25 protein, thereby carrying out auxiliary diagnosis of gastric cancer. The SMIM25 recombinant fusion protein can be used as an antigen for related immunological experiments; and the SMIM25 recombinant fusion protein is used as animmunogen to be mixed with an adjuvant and then injected into a rabbit, and the polyclonal antibody is obtained after protein purification, and the obtained polyclonal antibody can be combined with prokaryotic expression protein and the SMIM25 protein in human cells so as to be used for further researching the specific functions of the SMIM25 gene.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to a recombinant plasmid containing human SMIM25 gene, genetic engineering bacteria, recombinant protein, polyclonal antibody, preparation method and application. Background technique [0002] Malignant tumors have always been one of the major diseases faced by countries all over the world. Gastric cancer is one of them, and it is also one of the malignant tumors with the highest mortality rate in China. At present, there are not many markers for early diagnosis of the disease, and the specificity is not enough, which leads to the fact that some patients are already in the middle and late stage of the disease when they are diagnosed with gastric cancer. In addition, due to tumor recurrence and metastasis, the 5-year survival rate of patients with advanced gastric cancer is less than 30%. Therefore, further exploration of the molecular mechanism of gastric cancer proliferatio...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N1/21C07K19/00C07K16/18G01N33/574
CPCC07K14/47C07K16/18C07K2319/21C07K2319/23C07K2319/41C07K2319/42C12N15/70G01N33/57446
Inventor 林志娟梁淑娟付晓燕秦浩
Owner WEIFANG MEDICAL UNIV
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