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Expression vector and preparation method of HNTX (Hainantoxin)-IV analogue rHNIV-01

An expression vector, Hainan toxin technology, applied in the expression vector of Hainan toxin-IV analogue rHNIV-01 and its preparation field, can solve the problems of high technical threshold and low degree of product homogeneity, and achieve the effect of low-cost purification method

Inactive Publication Date: 2014-10-15
NAT UNIV OF DEFENSE TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the use of Pichia pastoris has the problem of low homogeneity of the product, and compared with E. coli preparation, there is a higher technical threshold
There is no report on the use of E. coli to prepare rHNIV-01 analogues

Method used

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  • Expression vector and preparation method of HNTX (Hainantoxin)-IV analogue rHNIV-01
  • Expression vector and preparation method of HNTX (Hainantoxin)-IV analogue rHNIV-01
  • Expression vector and preparation method of HNTX (Hainantoxin)-IV analogue rHNIV-01

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1 Construction and use of expression vector pWE-rHNIV-01

[0026] Using the pET-43a vector retained in our laboratory as a template, using the PCR-based seamless cloning method (for specific experimental methods, see Journal of Biochemical and Biophysical Methods67(1), 67–74.(2006)), the coding The sequence of GST (SEQ ID NO.1) replaced the original NusA sequence, and at the same time, the sequence encoding SUMO (SEQ ID NO.2) was inserted downstream of GST, and finally the sequence encoding rHNIV-01 (SEQ ID NO.3) was inserted Downstream of the ULP1 enzyme cleavage recognition site of SUMO ( figure 1 ). The PCR product was digested with DpnI and then transferred into E. coli DH5α. The plasmid was extracted and verified by DNA sequencing. Finally, the prokaryotic expression plasmid containing the expression cassette of GST+SUMO+rHNIV-01 was obtained and named pWE-rHNIV-01. The sequence is as shown in SEQ Shown in ID NO.4.

[0027] The primers used are as follow...

Embodiment 2

[0043] Example 2 Induced expression of expression plasmid pWE-rHNIV-01

[0044] The expression plasmid was transformed into Escherichia coli BL21(DE3) using the conventional calcium chloride transformation method, and then spread on 1 ampicillin-resistant (100 μg / mL) LB solid medium, and placed upside down at 37 degrees for 16 hours. The next day, pick 1-2 single colonies and inoculate them in 5 mL of ampicillin-resistant LB culture medium, and cultivate them at 37 degrees and 220 rpm until OD 600 To around 1 (this step is the enrichment of the seed medium). Transfer 5mL seed culture medium into fresh 500mL LB (100μg / mL ampicillin) culture solution, and continue to cultivate at 37°C and 220rpm until OD 600 to around 0.6, add IPTG with a final concentration of 0.5mM at this time to induce expression at 37 degrees, and the induction time is 4 hours. Through experiments, we found that the fusion protein containing rHNIV-01 can also be expressed normally in a partially soluble f...

Embodiment 3

[0045] Example 3 Preliminary separation and purification of rHNIV-01 fusion protein and protein kinase digestion

[0046] The thallus that has been induced in Example 2 was centrifuged at 4500 g for 10 minutes to obtain the thallus. Then add an appropriate volume of PBS buffer to resuspend the cells, followed by sonication. After the sonication is completed, centrifuge at 15,000 rpm for 10 minutes to obtain supernatant and precipitate. A dialysis membrane of appropriate length (molecular weight cut-off: 35 kDa) was used to dialyze the ultrasonic supernatant after centrifugation, and dialyze overnight at 4°C. After the dialysis is completed, the liquid in the dialysis bag is subjected to high-speed centrifugation, and the dialysis supernatant is taken for subsequent experiments. An appropriate dose of ULP1 kinase (to release rHNIV-01) was added to the supernatant and digested at 30°C for 4 hours.

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Abstract

The invention provides an expression vector and a preparation method thereof, and high-purity recombinant HNTX (Hainantoxin)-IV analogue rHNIV-01 with biological activity can be rapidly prepared by the expression vector. Compared with natural HNTX-IV, an amino acid sequence of rHNIV-01 is almost the same as that of natural HNTX-IV, and only the C terminal of rHNIV-01 is not subjected to amidation modification. The method does not adopt conventional affinity chromatography for purifying fusion protein, protein kinase is directly added in a cell lysis product for cutting, then a cutting product rHNIV-01 is directly subjected to selective active extraction with a TCA (trichloroacetic acid) extraction method, and finally, RP-HPLC (reversed phase high-performance liquid chromatography) is adopted for purification. With the adoption of the method, 3 mg / L high-purity rHNIV-01 can be obtained stably; and purified rHNIV-01 can selectively inhibit TTX-S (tetrodotoxin-sensitive) sodium current on DRG (dorsal root ganglia) cells of a rat.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to an expression vector of Hainan toxin-IV analogue rHNIV-01 and a preparation method thereof. Background technique [0002] Peptide drugs are more and more favored by major pharmaceutical companies due to their high selectivity and no accumulation in the body. In particular, polypeptide molecules containing multiple pairs of disulfide bonds have better stability and absorbability than linear polypeptide molecules, so they are a hot spot in drug design and development. Natural Hainan toxin-IV (HNTX-IV) is a neuropeptide toxoid isolated from the unique Hainan tarantula in my country. It contains 35 amino acids, the C-terminus has amidation modification, and contains three pairs of disulfide bonds, and the pairing of disulfide bonds is a typical ICK motif (Cys2-Cys17, Cys9-Cys24, Cys16-Cys31). Preliminary studies have shown that HNTX-IV can selectively and efficie...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12P21/02
Inventor 孟尔张东裔张辉鲍邵衡李文颖吴磊朱凌云柳珑
Owner NAT UNIV OF DEFENSE TECH
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