Method for acquiring human alpha fetoproteins

A technology of alpha-fetoprotein and human, applied in animal/human protein, pregnancy protein, mammalian protein, etc., to achieve high purity and good activity

Inactive Publication Date: 2016-07-06
HAINAN MEDICAL COLLEGE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] At present, there is no report on the method of obtaining

Method used

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  • Method for acquiring human alpha fetoproteins
  • Method for acquiring human alpha fetoproteins
  • Method for acquiring human alpha fetoproteins

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Embodiment 1 utilizes the method for human alpha-fetoprotein expressed by Sf9 insect cell eukaryotic and pFastBac1 vectors

[0029] 1. The human alpha-fetoprotein gene (AFP) and the pFastBac1 vector were ligated after double digestion with restriction enzymes, and the E.coliDH5α bacteria containing the vector pFastBac1-AFP were transferred to 100mL ampicillin-containing bacteria at a ratio of 1:100. culture medium with shaking at 37°C overnight, and the expression plasmid was extracted.

[0030] 2. Identify the correct recombinant plasmid pFastBac1-AFP and transform it into E.coliDH10Bac, transform the connected plasmid into DH10Bac cells, and spread it on the LB containing kanamycin, gentamicin, tetracycline, IPGT and X-gal combination On the medium plate (the five components are mixed in a ratio of 1:1.5:2:1:4, and the mixed solution is added to the LB medium in a volume ratio of 1:1000), the blue and white spots are screened, and the insects are obtained by using the...

Embodiment 2

[0037] Embodiment 2 The method of human alpha-fetoprotein expressed by Sf9 insect cell eukaryotic and pFastBacDual vector

[0038] 1. The human alpha-fetoprotein gene and the pFastBacDual vector were digested with restriction endonucleases and ligated, and the ligated vector was transformed into E.coliDH10Bac to obtain a Bacmid expressing human alpha-fetoprotein. The Bacmid was transfected into Sf9 insect cells .

[0039] 2. Before transfection, put the cells in the logarithmic growth phase at an approximate density of 2×10 6 Each / mL Sf9 insect cells were evenly placed on the plate, and 2 mL of insect cell culture medium was added to each well, and stood at 27°C for 1 hour to allow the cells to adhere to the wall. It was observed under a microscope that 80% of the cells covered the plate.

[0040] Prepare the following solutions at the same time, solution A: add 5 μL of recombinant AFP / Bac to 100 μL of SFX-Insect insect cell culture medium, and mix well. Solution B: For each...

Embodiment 3

[0044] Example 3 The method of human alpha-fetoprotein expressed by Sf21 insect cell eukaryotic and pFastBac1 vector

[0045] 1. Ligate the human alpha-fetoprotein gene and the pFastBac1 vector after double digestion with restriction enzymes, transform the ligated vector into E.coliDH10Bac, and obtain the Bacmid expressing human alpha-fetoprotein. Transfect the Bacmid into Sf21 insect cells .

[0046] 2. Before transfection, put the cells in the logarithmic growth phase at an approximate density of 1 to 3×10 6 Each / mL Sf21 insect cells were evenly placed on the plate, and 2 mL of insect cell culture medium was added to each well, and stood at 27°C for 1 hour to allow the cells to adhere to the wall. It was observed under a microscope that 80% of the cells covered the plate.

[0047] Prepare the following solutions at the same time, solution A: add 5-20 μL of recombinant AFP / Bac to 100 μL of SFX-Insect insect cell culture medium, and mix well. Solution B: For each transfectio...

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Abstract

The invention discloses a method for obtaining human alpha-fetoprotein, which comprises cloning the human alpha-fetoprotein gene into insect cell expression vectors pFastBac 1, pFastBac Dual or pFastBac1‑Gus, and transforming DH10Bac to obtain human alpha-fetoprotein-containing Bacmid gene, Bacmid transfected insect cells 2 to 4 days after the secretion of human alpha-fetoprotein into the culture medium, human alpha-fetoprotein secreted by nickel column and gel chromatographic column after purification can be active and extracted from human embryonic blood of alpha-fetoprotein. The invention utilizes the eukaryotic expression of Sf9 and Sf21 insect cells to obtain human alpha-fetoprotein with better activity.

Description

technical field [0001] The invention belongs to the field of biotechnology, relates to genetic engineering and protein engineering, in particular to a method for obtaining human alpha-fetoprotein. Background technique [0002] Alpha-fetoprotein (Alpha-fetoprotein, AFP) is a specific protein expressed in the early stage of liver cancer, and 70-80% of liver cancer patients have the characteristics of high expression of AFP gene during the onset of liver cancer. The AFP gene is open and expressed during fetal development, and 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. Therefore, AFP is used as the gold standard for the diagnosis of liver cancer. Clinically considered as a classic tumor marker for liver cancer. [0003] The sequence similarity rate of AFP and human serum albumin (HumanSerumAlbumin, HSA) is 60%. Although the sequence similarity rate of these two protein...

Claims

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Application Information

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IPC IPC(8): C07K14/47C12N15/85
CPCC07K14/4715
Inventor 林波刘坤朱明月李伟董栩李孟森
Owner HAINAN MEDICAL COLLEGE
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