Vitreoscilla hemoglobin mutant and gene and application thereof

A technology of hemoglobin and hyaline bacteria, applied in the field of genetic engineering, can solve problems that are not necessarily optimal

Active Publication Date: 2008-03-05
ZHEJIANG HISUN PHARMA CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, VHb is a prokaryotic protein that only exists in Vitreum hyaline. When its gene is transferred into heterologous cells, it faces a series of problems such as recognition, processi...

Method used

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  • Vitreoscilla hemoglobin mutant and gene and application thereof
  • Vitreoscilla hemoglobin mutant and gene and application thereof
  • Vitreoscilla hemoglobin mutant and gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1 Construction of cloning and expression vectors

[0035] Utilize the Vector NTI10 software of Invitrogen Company, design primer VH1 and VH2 according to the vgb of 1.4kb and its promoter sequence reported in literature Dikshit KL, WebsterDA.Gene, 1988,70:377-386:

[0036] VH1:5'-GC TCTAGA TTATTCAACCGCT

[0037] wxya

[0038] VH2: 5'-CG GAATTC TATGTTAGACCAGCAAAACCA-3' (SEQ ID No. 4)

[0039] EcoR I

[0040] Using the genome of Vitreoscilla filiformis ATCC43191 as a template, VH1 and VH2 were used as primers to amplify the vgb structural gene. The reaction conditions were: 94°C for 5 minutes, 94°C for 30s, 57°C for 30s, and 72°C for 40s. After 30 cycles, Extend at 72°C for 5 min. The PCR product was separated by 1% agarose gel electrophoresis, and a fragment around 500 bp was recovered. After being digested by EcoR I and XbaI, it was connected into pUC18 which had been digested by the same double enzyme to obtain pUC18vgb.

[0041] Also design primers Pvg...

Embodiment 2

[0050] Embodiment 2 utilizes error-prone PCR to obtain vgb gene shuffling material

[0051] Use pYG857 template, VH1 and VH2 as primers to amplify vgb, add MgCl to the PCR system 2 and MnCl 2 , so that Mg 2+ and Mn 2+ The final concentrations were 7.5mmol / L and 0.5mmol / L respectively, and the PCR reaction conditions and product processing were as described in Example 1. The error-prone PCR product was recovered by electrophoresis (Fig. 2), and double-digested with EcoR I and Xba[, and the digested product was connected to pYG857 through the same digestion to obtain the mutant gene vector pYG857EP, which was transformed into E.coli DH5α competent cells, Spread on LB agar plate containing ampicillin and culture overnight at 37°C.

[0052] Randomly pick a single transformant colony from the transformation plate, insert it into a 15ml test tube containing 5ml LB liquid medium, add antibiotics, seal the test tube mouth with a rubber cap, and cultivate at 37°C, 230r / min for abou...

Embodiment 3

[0053] Example 3 DNA shuffling of vgb gene

[0054] (1) DNA fragmentation reaction

[0055] Using pYG857 and 4 pYG857EP screened by error-prone PCR as templates, use the conditions described in Example 1 to amplify the original gene vgb and mutant gene vgb', recover the target fragment by electrophoresis, and dissolve it in an appropriate volume of ddH 2 O middle. In a 20 μl system, add about 0.5 μg of each of the five recovered products and 0.5 UDNase I, react at 25°C for 14-20min, and inactivate the enzyme at 90°C for 10min. The degree of fragmentation of the product was detected by agarose gel electrophoresis. It can be seen from Fig. 3 that when the digestion reaction is carried out for about 18 minutes, ideal fragments of 50 bp can be obtained. Cut out the gel block where the fragments are concentrated near 50bp, and recover it with the small fragment DNA rapid recovery kit.

[0056] (2) PCR without primers

[0057] Take 40 μl of small fragment recovery products dire...

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Abstract

The present invention discloses mutational Vitreoscilla hemoglobin gene sequence and its coded Vitreoscilla hemoglobin mutant, as well as the transformant obtained through transforming heterogenous host with the mutational Vitreoscilla hemoglobin gene and its application in fermentation. The mutational Vitreoscilla hemoglobin gene consists of 438 nucleotides and codes one segment of protein comprising 146 amino acids. The gene expressed protein can raise the biomass of the host cell in the condition of insufficient environmental oxygen dissolution, so that it may be applied for promoting the growth of the host cell in fermentation industry to raise the yield of the target product and lower production cost.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a Vitreoscilla hemoglobin mutant and its gene, an expression vector containing the gene, a transformant obtained by transforming the gene into a heterologous host cell, and its application in the fermentation industry application. Background technique [0002] Vitreoscilla is an obligate aerobic filamentous Gram-negative bacterium that can be isolated from ponds or rotting vegetables. As an obligate aerobic bacteria, Vitreoscilla can survive in an oxygen-poor environment because it can synthesize Vitreoscilla hemoglobin (VHb for short, the coding gene is called vgb or vhb, Genbank number M30794) for the sake. This protein is expressed in heterologous microorganisms, plants and even animal cells, and it is found that it can promote the growth of host cells under oxygen-limited conditions, increase the production of antibiotics, enzymes or other economic products, reduce produc...

Claims

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Application Information

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IPC IPC(8): C12N15/31C07K14/195C12N15/63C12N1/21
Inventor 朱春宝袁宁胡又佳朱宝泉
Owner ZHEJIANG HISUN PHARMA CO LTD
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