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Recombinant expression vector system, recombinant engineering bacterium and preparation method and application of recombinant expression vector system or recombinant engineering bacterium

A technology of recombinant engineering bacteria and expression vectors, applied in the field of bioengineering, can solve the problems of high cost, complicated and cumbersome HPV antibody process, etc.

Pending Publication Date: 2021-03-26
重庆博唯佰泰生物制药有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] In order to achieve the above purpose and other related purposes, the present invention provides a recombinant expression vector system, recombinant engineering bacteria and its preparation method and application, which are used to solve the complex, cumbersome, high cost and poor controllability of the HPV antibody preparation process in the prior art. The problem

Method used

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  • Recombinant expression vector system, recombinant engineering bacterium and preparation method and application of recombinant expression vector system or recombinant engineering bacterium
  • Recombinant expression vector system, recombinant engineering bacterium and preparation method and application of recombinant expression vector system or recombinant engineering bacterium
  • Recombinant expression vector system, recombinant engineering bacterium and preparation method and application of recombinant expression vector system or recombinant engineering bacterium

Examples

Experimental program
Comparison scheme
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Embodiment 1

[0109] Example 1 Construction and Transformation of Yeast Double Expression Plasmids

[0110] In order to simultaneously express HPV6 L1, HPV11 L1, HPV16 L1 and HPV 18L1 in yeast, two expression plasmids that can express two proteins at the same time need to be transformed, and each plasmid contains two independent expression cassettes. The screening markers contained are different, so that two different double expression plasmids can be introduced into yeast through two transformation and screening methods. The specific plasmid construction scheme is as follows:

[0111] 1.1 Construction of yeast double expression plasmid pMMZ

[0112] By PCR, the sequences shown in SEQ ID NO: 7-8 are used as primers, and the whole genome DNA of Hansenula spp. is used as a template to amplify the MOX promoter insert fragment (SEQ ID NO: 26); using SEQ ID NO: 10, The sequence shown in SEQ ID NO: 12 is used as a primer, and the pPICZB plasmid is used as a template to amplify a fragment contai...

Embodiment 2

[0166] Example 2 Construction of 6L1-11L1-pMMG double expression plasmid and 16L1-18L1-pMMZ double expression plasmid

[0167] 2.1 HPVL1 double expression plasmid construction

[0168] DNA sequences encoding HPV6 L1, HPV11 L1, HPV16 L1, and HPV18 L1, such as SEQ ID NO: 1-4, were synthesized by Suzhou Jinweizhi Co. The cloned plasmid pUC57 was used to obtain HPV6L1-pUC57, HPV11L1-pUC57, HPV16L1-pUC57, and HPV18L1-pUC57, respectively. Insert HPV6 L1 and HPV11 L1 into pMMG plasmid, and insert HPV16 L1 and HPV18 L1 genes into pMMZ plasmid, respectively.

[0169] 2.2 Construction of HPV6 L1 / HPV11 L1 double expression plasmid

[0170] By means of PCR, using the sequences shown in SEQ ID NO:19-20 as primers and the HPV11L1-pUC57 plasmid as a template, the HPV11 L1 fragment (SEQ ID NO:38) was amplified; BstBI+SbfI combined enzyme digestion to treat the HPV11 L1 fragment and pMMG plasmid, the two fragments were ligated to obtain 11L1-pMMG (SEQ ID NO: 39).

[0171] By means of PCR, ...

Embodiment 3 4

[0206] Example 3 Construction and screening of four expression strains

[0207] Based on the 6L1-11L1-pMMG double-expression plasmid and the 16L1-18L1-pMMZ double-expression plasmid constructed in Example 2, the two double-expression plasmids were integrated into the Han The host bacterium CBS4732 (ATCC 34438) of Sylvia sinensis was used to obtain recombinant engineering yeast strains expressing HPV6 L1, HPV11 L1, HPV16 L1, and HPV18 L1 at the same time. The specific steps are as follows:

[0208] 3.1 Construction and screening of HPV16 L1 / HPV18 L1 dual expression strains

[0209] The recombinantly constructed 16L1-18L1-pMMZ plasmid was linearized by BglII enzyme, and then electroporated into Hansenula CBS4732. The electroporation conditions were 1500V, 120Ω, 50μF. After electroporation, the bacterial solution was coated on a YPD plate (200 μg / ml Zeocin), and incubated overnight at 37°C. Some clones were picked and streaked on a YPD plate (1500 μg / ml Zeocin), cultured upside...

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Abstract

The invention provides a recombinant expression vector system, a recombinant engineering bacterium and a preparation method and application of the recombinant expression vector system or the recombinant engineering bacterium. The recombinant expression vector system at least comprises two recombinant expression vectors; each recombinant expression vector includes one or more nucleic acid expression cassettes; each nucleic acid expression cassette includes a target gene, and a promoter operably linked to the target gene; each target gene is independently selected from one of HPV6 L1, HPV11 L1,HPV16 L1, HPV18 L1, HPV31 L1, HPV33 L1, HPV35 L1, HPV39 L1, HPV45 L1, HPV51 L1, HPV52 L1, HPV53 L1, HPV56 L1, HPV58 L1, HPV59 L1, HPV66 and HPV68 L1, and all the target genes are different from each other. The recombinant engineering bacterium can be obtained by converting the recombinant expression vector system into an engineering bacterium. The recombinant engineering bacterium can express various HPV L1 proteins at the same time, and is used for preparing HPV vaccines.

Description

technical field [0001] The invention relates to a bioengineering technology, in particular to a recombinant expression vector system, a recombinant engineered bacterium and a preparation method and use thereof. Background technique [0002] Human papillomavirus (HPV) mainly infects human epithelial cells and causes damage to epithelial cells, leading to a series of diseases including genital warts and cervical cancer. In recent years, more and more studies have shown that HPV infection can also cause other diseases. Anal and genital-related cancers, such as vaginal cancer, anal cancer, penile cancer, etc., are even related to oropharyngeal cancer. Among them, cervical cancer is the most concerned and the most clear pathogenic mechanism of HPV. As the third largest female cancer in the world, cervical cancer kills 250,000 people every year, and more than 99% of cervical cancers are caused by HPV infection. [0003] The main route of transmission of HPV is sexual transmission...

Claims

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Application Information

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IPC IPC(8): C12N15/81C07K14/025
CPCC07K14/005C12N15/81C12N15/815C12N2710/20022
Inventor 丁珊潘婷姬美彤
Owner 重庆博唯佰泰生物制药有限公司
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