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Method for efficiently expressing AFP3-PTEN fusion protein

A technology of AFP3-PTEN and fusion protein, applied in the direction of pregnancy protein, mammalian protein, chemical instruments and methods, etc.

Pending Publication Date: 2020-10-23
HAINAN MEDICAL COLLEGE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, there is no report on the method of high-efficiency expression and purification of fusion protein AFP3-PTEN by using liver cancer cells

Method used

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  • Method for efficiently expressing AFP3-PTEN fusion protein
  • Method for efficiently expressing AFP3-PTEN fusion protein
  • Method for efficiently expressing AFP3-PTEN fusion protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Embodiment 1 Utilizes the method of high-efficiency expression and purification of AFP3-PTEN fusion protein by HLE cells

[0027] 1. Establishment of HLE cells expressing HBx

[0028] According to the human HBx gene sequence (NCBI accession number is AB210819), the synthetic gene was double-digested with Xba I and Xho I, connected to the expression vector PTT5 (Invitrogen) with T4 ligase, transformed into DH5α cells, extracted the plasmid, and then transfected into HLE Cells were cultured for 48 hours to detect the expression of HBx protein.

[0029] 2. The plasmid containing PTT5-AFP3-PTEN was transfected into HLE cells expressing HBx, cultured for 48 hours, and the culture medium was collected.

[0030] 3. Separation, purification and identification of AFP3-PTEN fusion protein. Since the protein is secreted, the medium and cell mixture was centrifuged at 8000r / min for 5min, and the supernatant was concentrated and switched through a tangential flow membrane. The sol...

Embodiment 2

[0033] Embodiment 2 Utilizing HepG2 cells to efficiently express and purify the method of AFP3-PTEN fusion protein

[0034] 1. Establishment of HepG2 cells expressing HBx

[0035] According to the human HBx gene sequence (NCBI accession number is AB210819), the synthetic gene was double-digested with Xba I and Xho I, connected to the expression vector PTT5 (Invitrogen) with T4 ligase, transformed into DH5α cells, extracted the plasmid, and then transfected into HepG2 Cells were cultured for 48 hours to detect the expression of HBx protein.

[0036] 2. The plasmid containing PTT5-AFP3-PTEN was transfected into HepG2 cells expressing HBx, cultured for 96 hours, and the medium was collected.

[0037] 3. Separation, purification and identification of recombinant proteins. After the medium and cell mixture was centrifuged at 8000r / min for 5min, the supernatant was taken to concentrate the switching solution through a tangential flow membrane, and the solution was HBS buffer (10mM...

Embodiment 3

[0040] Embodiment 3 Utilizing Bel-7402 cells to efficiently express and purify the method of AFP3-PTEN fusion protein

[0041] 1. Establishment of Bel-7402 cells expressing HBx

[0042] According to the human HBx gene sequence (NCBI accession number is AB210819), the synthetic gene was digested with Xba I and Xho I, connected to the expression vector PTT5 (Invitrogen) with T4 ligase, transformed into DH5α cells, extracted the plasmid, and then transfected into Bel -7402 cells were cultured for 72 hours to detect the expression of HBx protein.

[0043] 2. The plasmid containing PTT5-AFP3-PTEN was transfected into Bel-7402 cells expressing HBx, cultured for 85 hours, and the medium was collected.

[0044] 3. Separation, purification and identification of recombinant proteins. Since the protein is secreted and expressed, the culture medium and the cell mixture were centrifuged at 8000r / min for 5min, and the supernatant was lyophilized and replaced with HBS (10mM Hepes, pH 7.2, ...

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Abstract

The invention discloses a method for efficiently expressing an AFP3-PTEN fusion protein, and belongs to the technical field of biology. The method comprises the steps of cloning an HBx gene to a constructed expression vector, enabling the constructed vector to be transfected with human liver cancer cells, after expressing HBx proteins in the liver cancer cells, enabling the third structured domainof AFP and an overall-length PTEN gene to be connected to the expression vector and transfected into the human liver cancer cells expressing the HBx proteins, expressing the HBx proteins in the livercancer cells, promoting efficient secretory expression of a human AFP3-PTEN fusion protein through the HBx proteins, and concentrating and purifying culture mediums to obtain the AFP3-PTEN fusion protein. The AFP3-PTEN fusion protein expressed by the liver cancer cells can restrain cell proliferation and can enable the cell proliferation capacity of NIH 3T3 cells to be reduced by 15%-60%. But a reference protein AFP enables cell proliferation capacity to be obviously increased by 22-125%. Serum proteins do not have notable capacity for promoting NIH 3T3 cell proliferation. The obtained AFP3-PTEN fusion protein can be used for researching tumor treatment.

Description

technical field [0001] The invention belongs to the field of biotechnology, relates to genetic engineering and protein engineering, in particular to the fusion expression of the third structural domain of AFP (human alpha-fetoprotein) and PTEN (the phosphatase and tensin homologous gene missing from human chromosome 10) and purification methods. Background technique [0002] The Alpha-fetoprotein (AFP) gene is open and expressed during fetal development, and it is basically closed after two years of birth. However, when liver cancer occurs in adults, the AFP gene is reactivated and expressed in large quantities, so AFP is used As a standard for diagnosing liver cancer, AFP can enter cancer cells through its receptors. Therefore, AFP can also be used as a carrier of targeted drugs, carrying anticancer drugs and the like into cancer cells, thereby killing cancer cells. [0003] PTEN protein can inhibit the progress of tumor by antagonizing the activity of tyrosine kinase and...

Claims

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Application Information

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IPC IPC(8): C12N15/85C07K19/00
CPCC12N15/85C07K14/4715C07K14/4703C12N9/16C12Y301/03067C07K2319/21C12N2800/107
Inventor 林波朱明月李伟董栩刘坤李孟森
Owner HAINAN MEDICAL COLLEGE
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