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Soluble aquaporin AqpZ fusion vector and method for constructing same

An aquaporin, soluble technology, applied in the biological field, can solve problems such as uncertainty and achieve the effect of eliminating influence

Inactive Publication Date: 2018-07-10
NANJING UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the use of nano-phospholipid discs requires the participation of lipids, which introduces a certain degree of uncertainty.

Method used

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  • Soluble aquaporin AqpZ fusion vector and method for constructing same
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  • Soluble aquaporin AqpZ fusion vector and method for constructing same

Examples

Experimental program
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Effect test

Embodiment 1

[0034] 1. A construction method of the maltose-binding protein carrier pET28a-ΔMBP without the N-terminal signal peptide:

[0035] a) Design a pair of primers based on the pMBP-p plasmid purchased from Wuhan Miaoling Biotechnology. The oligonucleotide sequence upstream of the primer is shown in SEQ ID No.1, and the oligonucleotide sequence downstream of the primer is shown in SEQ ID No.2. Using the pMBP-p plasmid as a template, the cDNA of the maltose-binding protein with the N-terminal signal peptide removed was obtained through PCR reaction. The PCR reaction system configuration is shown in Table 1.

[0036] Table 1 PCR reaction system preparation table

[0037]

[0038]

[0039] PCR results such as figure 1 shown. The first lane is Marker, the second lane is the target fragment ΔMBPcDNA, and the length of the target fragment is 1032bp.

[0040] b) Recover the maltose binding protein cDNA that removes the N-terminal signal peptide, use NcoI and NdeI to double dige...

Embodiment 2

[0056] Example 2: Expression method of a soluble aquaporin AqpZ vector

[0057] Transform the correctly identified pET28a-ΔMBP-AqpZ-ApoAI*-His plasmid into E. coli expression host BL21(DE) to obtain a single colony of stable transformation; inoculate a single colony into LB liquid medium containing 50 μg / ml kanamycin In 200 rpm, 37 ° C overnight culture, to obtain overnight bacteria. Inoculate overnight bacteria into TB medium at an inoculation ratio of 1:200, and culture to OD at 230 rpm at 30°C 600 After reaching 0.8-1.0, add IPTG (isopropylthiogalactopyranoside) with a final concentration of 0.1 mM, and then culture at 16°C for 18 hours. The cells were collected by centrifugation at 4000 rpm for 15 minutes. The bacteria were resuspended in PBS buffer, and then centrifuged at 4000 rpm for 15 minutes to obtain soluble fusion expression of aquaporin ΔMBP-AqpZ-ApoAI*-His bacteria.

Embodiment 3

[0058] Example 3: Verification of a soluble aquaporin AqpZ carrier

[0059] Step 1, sonicate the fusion protein ΔMBP-AqpZ-ApoAI*-His cells obtained in Example 2. The ultrasonic time is 15 minutes, the ultrasonic is 2 seconds, the interval is 4 seconds, and the ultrasonic power is 60W. Finally, centrifuge the sonicated bacteria at 10,000g at 4°C for 45 minutes, collect the supernatant to obtain the whole protein fraction; centrifuge the supernatant at 100,000g at 4°C for 2 hours, collect the supernatant to obtain the soluble fraction point.

[0060] Step 2: Collect the whole protein fraction and soluble fraction of the fusion protein, take 10 μL sample, add 10 μL 2*loading buffer, incubate at 75°C for 10 minutes, after cooling, centrifuge at 12,000g for 5 minutes, and apply 15 μL of sample; first Run glue at 90 volts for 30 minutes, then change to 150 volts and run glue for 50 minutes.

[0061] Step 3, use nitrocellulose membrane for wet transfer, the transfer condition is c...

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Abstract

The invention discloses a soluble aquaporin AqpZ fusion vector and a method for constructing the same. Maltose binding proteins delta MBP without signal peptides are used as N-terminal hydrophobic tags, truncated human apolipoproteins ApoAI* are used as C-terminal amphiphilic tags, C-terminal His (histidine) is used as a purification and detection tag, and accordingly the pET28a-delta MBP-AqpZ-ApoAI*-His recombinant vector can be constructed. The soluble aquaporin AqpZ fusion vector and the method have the advantages that originally fat-soluble aquaporins become water-soluble and can be dissolved in water phase without the aid of detergents, the expression quantities of the aquaporins can be obviously increased, and the soluble aquaporin AqpZ fusion vector and the method are applicable toindustrial production.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a soluble aquaporin AqpZ fusion carrier and a construction method thereof. Background technique [0002] As an important class of channel proteins, aquaporins play a key role in maintaining the balance of intracellular and intracellular osmotic pressure. Understanding its structure can help researchers better elucidate how aquaporins work. Due to the low abundance of aquaporins, it is difficult for researchers to obtain a sufficient amount of samples for subsequent analysis; not only that, aquaporins are very hydrophobic, so they must be dissolved from the membrane environment with the help of detergents. In the water environment, the introduction of detergents will change the natural conformation of aquaporins to a certain extent, causing difficulties in analysis. [0003] The literature "Applied Microbiology and Biotechnology March 2009, Volume 82, Issue 3, pp 463–470...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C07K19/00
CPCC07K14/245C07K14/775C07K2319/00C07K2319/21C07K2319/24C12N15/62C12N15/70
Inventor 周敏洪涛王坤卢颖洪李霖
Owner NANJING UNIV OF SCI & TECH
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