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Method for preparing gene recombinant human ferritin light chain

A technology of gene recombination and ferritin, applied in the field of bioengineering, can solve the problems of difficult comparison of consistency and achieve the effect of improving accuracy

Inactive Publication Date: 2010-12-22
张曼
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In particular, there are a variety of ferritin detection methods currently used clinically, and the consistency between various methods is difficult to compare

Method used

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  • Method for preparing gene recombinant human ferritin light chain
  • Method for preparing gene recombinant human ferritin light chain

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Experimental program
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Effect test

Embodiment 1

[0021] Preparation of embodiment 1 subcloning plasmid pGEM-T / ferritin L

[0022] Total RNA was extracted from human fresh whole blood cells with Trisol kit, and the cDNA fragment of human ferritin light chain gene (Ferritin L) was obtained from human fresh whole blood cells by RT-PCR technology. The primers used were:

[0023] Upstream primer: 5'-CATATGAGCTCCCAGATTCGTCAG-3';

[0024] Downstream primer: 5'-GGATCCTCTTAGTCGTGCTTGAGAG-3'

[0025] After the PCR product was recovered and purified, it was ligated with the pGEM-T vector overnight at 4°C, transformed into competent Top10 bacteria, inoculated into LB culture dishes containing ampicillin, screened for positive clones, and sequenced for identification. The bacterial strain is Escherichia coli containing the Ferritin L coding sequence plasmid pGEM-T / ferritin L.

Embodiment 2

[0026] Example 2 Construction of expression vector pET-30a / ferritin L

[0027] Using the plasmid pGEM-T / FTL as a template, double digestion was performed with NdeI and BamHI; at the same time, the empty plasmid pET-30a was double digested with NdeI+BamHI. Recover the above-mentioned digested products separately, mix them in an appropriate ratio, connect them overnight at 16°C with T4 ligase, transform competent BL21(DE3) bacteria, inoculate LB culture dishes containing kanamycin, and screen for positive clones.

[0028] The plasmid pET-30a / ferritin L of the positive clone was extracted for sequencing identification. The DNA sequence of the insert fragment is completely consistent with the ferritin L sequence encoding 175 amino acid residues in GenBank (number in GenBank), and the reading frame is correct, which can encode the desired target protein. Such as figure 1 shown.

Embodiment 3

[0029] Induced expression of embodiment 3 Ferritin L protein

[0030] The engineered bacteria BL21 (DE3) containing pET-30a / ferritin L plasmid was inoculated in 5ml of LB medium containing kanamycin (50ml), cultivated overnight, and transferred to 1L LB medium at 1:50 the next day ( Kanamycin resistance), 37°C, 240r / min rotation culture for about 2h, make A600 reach 0.6-0.8, add inducer IPTG to the final concentration of 1mmol / L, continue to shake under the above conditions for 4h. After the cultivation, the bacterial pellet was collected by centrifugation at 8000 g for 10 min. Wash 3 times with PBS, lyse the cells by ultrasonic in ice bath, centrifuge at 12000g for 20min, separate supernatant and precipitate.

[0031] The expressed protein of pET-30a / ferritin L was analyzed by SDS-PAGE, such as figure 2 As shown, the expressed protein with a relative molecular mass of about 20,000 can be seen, which is consistent with the expected size. After ultrasonic cracking, it was f...

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Abstract

The invention discloses a method for preparing a gene recombinant human ferritin light chain. The method comprises the following steps of: amplifying a coding genetic sequence of a ferritin light chain (Ferritin L) in human fresh complete blood cells by PCR, cloning the amplified coding genetic sequence into an expression vector pET-30a by enzyme digestion and connection, transforming E.coli BL21 (DE3), identifying a positive cloning colony, performing induction expression on the Ferritin L by IPTG, and analyzing the antigenicity of the Ferritin L by using Westernblot. The method successfully constructs the prokaryotic expression vector of the FTL, and obtains the pure ferritin light chain by induction expression and purification in colon bacillus. Proved by the Westernblot, the protein has good antigenicity.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a preparation method of genetically recombined human ferritin light chain. Background technique [0002] The development of clinical medicine has higher and higher requirements for the accuracy, stability and sensitivity of test results, so high-quality quality control products, calibrators and reference products are key products in test and diagnosis. At present, my country basically uses the first-generation and second-generation tissue-purified ferritin products, and their activity, linearity, stability, specificity, sensitivity, and storage time vary greatly due to the uncertainty of the source. Therefore, genetic recombination methods are rapidly applied and promote such products into the third generation. Due to the complex structure of ferritin, the common renaturation of 24 subunit units is extremely difficult, and it is very technical to obtain the st...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12P21/02C12R1/19
Inventor 张曼
Owner 张曼
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