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DNA sequence, recombinant vector, single and double auxotrophic Hansenula polymorpha, and preparation method thereof

A technology of Hansenula polymorpha and auxotrophy, applied in the field of genetic engineering, can solve the problems of high drug resistance and false positive ratio, difficulty in obtaining transformants, unclear mutation sites, etc., and achieve convenient source of strains. , The effect of clear mutation mechanism and low reversion mutation rate

Inactive Publication Date: 2011-11-09
BEIJING INST OF BIOLOGICAL PROD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Hansenula, as a host for the expression of various exogenous genes, requires many different types of screening markers, which mainly include two types: one is auxotrophic selection markers, such as URA3, LEU2, TRP3, HIS4, ADE11, LYS and other marker genes, the corresponding gene deletion host bacteria can only grow in the complete medium, but can not grow in the basal medium lacking the corresponding nutrients, after the carrier DNA with the screening marker is integrated with the host chromosome , so that the host bacterium can grow in the selection medium; the other is a dominant selection marker, such as anti-G418, anti-humic acid or anti-Zeocin gene, etc. Although this type of selection marker is simple and convenient to use, its existence (such as ) has many obvious disadvantages such as expensive, easy to produce drug resistance and high false positive rate
Most of the current methods for obtaining auxotrophic Hansenula spp. are to obtain corresponding selection markers through traditional methods such as mutagenesis, hybridization, or protolipid fusion. However, these methods have many outstanding shortcomings, such as relatively low genetic stability. Low, there are multiple mutation effects, the mutation site is not clear, it is difficult to obtain ideal transformants, etc.

Method used

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  • DNA sequence, recombinant vector, single and double auxotrophic Hansenula polymorpha, and preparation method thereof
  • DNA sequence, recombinant vector, single and double auxotrophic Hansenula polymorpha, and preparation method thereof
  • DNA sequence, recombinant vector, single and double auxotrophic Hansenula polymorpha, and preparation method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0097] The construction of the recombinant plasmid p18T-KURA3 of embodiment 1 inactivation URA3 gene

[0098] Such as figure 1 As shown, the construction process of the recombinant plasmid p18T-KURA3 is as follows. The primers were designed according to the reported (GenBank) nucleotide sequence of URA3 of Hansenula polymorpha, as shown in Table 1.

[0099] Table 1

[0100]

[0101] Extract the total DNA of Hansenula polymorpha ATCC 26012 as a template, use URA3-F and URA3-B as primers, and amplify the URA3 gene sequence according to the following PCR reaction system:

[0102] Template DNA 2μl

[0103] URA3-F 1μl

[0104] URA3-B 1μl

[0105] dNTP 5μl

[0106] 10*PCR ExTaq buffer 5μl

[0107] ExTaq enzyme 1μl

[0108] Deionized water 35μl

[0109]

[0110] Total volume 50μl

[0111] Wherein, the reaction condition of carrying out PCR reaction on PCR instrument is as follows:

[0112] 94°C, 5 minutes; then cycle 30 times at 94°C, 40 se...

Embodiment 2

[0134] Example 2 Construction of the recombinant plasmid p18T-KLEU2 containing the inactivated LEU2 gene

[0135] Such as figure 2 As shown, the construction process of the recombinant plasmid P18T-KLEU2 is as follows.

[0136] Primers were designed according to the reported (GenBank) nucleotide sequence of Hansenula polymorpha LEU2, see Table 2.

[0137] Table 2

[0138]

[0139] The total DNA of Hansenula polymorpha ATCC 26012 was extracted as a template, and a segment of upstream DNA sequence of LEU2 gene, namely L1 fragment, was amplified with LEU2-F1 and LEU2-B1 as primers, with a length of 525 bp. LEU2-F2 and LEU2-B2 were used as primers to amplify a downstream DNA sequence of the LEU2 gene, namely the L2 fragment, with a length of 570 bp.

[0140] Proceed according to the following PCR reaction system, wherein primer 3 is LEU2-F1, primer 4 is LEU2-B1 or primer 3 is LEU2-F2, primer 4 is LEU2-B2:

[0141] Template DNA 2μl

[0142] Primer 31μl

[0143] Primer 41μ...

Embodiment 3

[0153] Example 3 Construction and identification of uracil monoauxotrophic Hansenula polymorpha

[0154] The p18T-KURA3 constructed in Example 1 was transformed into Hansenula polymorpha ATCC 26012 by electroporation, and the uracil monoauxotrophic Hansenula strain was screened and identified. The specific steps were as follows:

[0155] 1. Preparation of competent Hansenula polymorpha ATCC 26012

[0156] Hansenula polymorpha ATCC 26012 was streak-inoculated on the slant medium (it is the YPD medium containing 7.5% agar) for activation, and the monoclonal colony was picked and inoculated in 2ml of YPD liquid medium with an inoculation loop , at 37°C in a shaker at 250rpm for 24 hours, then inoculate 2ml of the bacterial liquid into 50ml of YPD liquid medium, and cultivate it in a shaker at 250rpm at 37°C until the logarithmic growth phase (OD 600 Between 1.2-1.5), centrifuge at 3000rpm for 10 minutes and collect the bacteria; resuspend with potassium phosphate buffer of pH 7....

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Abstract

The invention relates to a double auxotrophic yeast, wherein the yeast is Hansenula polymorpha, and the orotic glycoside-5-phosphate decarboxylase gene and the beta-isopropyl malate dehydrogenase gene of the Hansenula polymorpha are blocked. The invention also relates to a preparation method of the double auxotrophic yeast. In addition, the invention also provides a DNA sequence and a recombinant vector used to prepared the double auxotrophic yeast. The double auxotrophic yeast of the invention has the advantages of low reverse mutation, high genetic stability, high biomass, and the like, and plays an important role in genetic engineering vaccine production; for example, HPV16-type L1 and 58L2 protein have double expression with high efficiency in the yeast.

Description

technical field [0001] The present invention relates to the field of genetic engineering, in particular, the present invention relates to a DNA sequence, a recombinant vector containing the DNA sequence, a single auxotrophic Hansenula polymorpha, a uracil and leucine double auxotrophic polymorphic Hansenula and its preparation method. Background technique [0002] Yeast, as a lower unicellular eukaryote, not only has the advantages of fast growth and simple genetic manipulation of prokaryotes, but also has the advantages of gene expression regulation mechanism and protein post-translational modification mechanism of higher eukaryotes. At present, the use of yeast as an expression system has been widely used and recognized. As the first expression system capable of expressing eukaryotic genes, Saccharomyces cerevisiae has successfully expressed a large number of proteins. However, there are many shortcomings in the application of this expression system, such as the lack of s...

Claims

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Application Information

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IPC IPC(8): C12N1/16C12N15/11C12N15/63C12R1/78
Inventor 李启明梁宇靳玉琴张靖陈实张学峰马智静于洁卫江波沈心亮
Owner BEIJING INST OF BIOLOGICAL PROD
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