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Yeast proliferation model of human papilloma virus as well as constructing method and uses

A technology for human papillomavirus and yeast, which is applied in the direction of introducing foreign genetic material, fungi, recombinant DNA technology using vectors, etc., can solve the problems of cumbersome methods, low virus yield, and difficult operation, and achieves improved yield and milder operation. , the effect of improving the efficiency of DNA circularization

Inactive Publication Date: 2008-06-18
SHANDONG UNIV
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  • Abstract
  • Description
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Problems solved by technology

Kreider et al. (1987) established a raft (raft) culture system, but the disadvantage is that the method of this culture system is very cumbersome, the operation is difficult, and the virus yield is very low
After searching, there are no reports at home and abroad on the establishment of a yeast proliferation model of human papillomavirus (HPV58) and its application using high-risk HPV58 as the research object

Method used

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  • Yeast proliferation model of human papilloma virus as well as constructing method and uses
  • Yeast proliferation model of human papilloma virus as well as constructing method and uses
  • Yeast proliferation model of human papilloma virus as well as constructing method and uses

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Embodiment

[0047] 1. Construction of the HPV genome (HPV-Ura3) containing yeast auxotrophy screening marker Ura3 This method is also applicable to inserting Ura3 into the viral genome containing the mutant gene

[0048] 1. Using the pRS316 plasmid containing Ura3 as a template, carry out PCR amplification with primers containing SgrA I restriction site, and the amplified product is the target gene (Ura3) of about 1.1Kbp.

[0049] Upstream primer: 5′-GAT CCA CCG GCG GCA GAT TGT ACT GAG AGT G-3′

[0050] Downstream primer: 5′-CTA GGC GGC CAC TAG TAT ACA TGC ATT TAC-3′

[0051] 2. The above PCR products were gel-cut and recovered. The recovered PCR product was connected to the pMD19-T simple vector to be pMD19-T-Ura3, and transformed into E.coli Competent Cell DH5α by heat, and the cells were cultured overnight on a plate.

[0052] 3. The pMD19-T-Ura3 with correct sequencing was digested with SgrAI; at the same time, pEGFPN1-HPV58 was digested with SgrAI (cutting point is located on the L...

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Abstract

The present invention discloses a human papilloma virus cerevisiae proliferation model and a preparation method, which is the saccharomyces cerevisiae cell Y303 ATCC No.96659 or saccharomyces cerevisiae cell YPH500 ATCC No.76626 of a HPV genome containing a cerevisiae nutrition defect screening mark Ura3. The model of the present invention can be used for researching on the function of HPV gene, or a cerevisiae RNA interfere technology is used for researching on the functions of different genes, or a cerevisiae expression vector is introduced again to express interested foreign protein in cells, so as to research on the effect of the cerevisiae expression vector in virus living period, or virus main capsid protein L1 expression vector is introduced for packing infective virus granules to be used for the experiment or research on HPV bacterin.

Description

technical field [0001] The present invention relates to a human papillomavirus yeast proliferation system and its establishment method and application; in particular, it relates to a human papillomavirus (HPV58 type) yeast proliferation model and its establishment method and application. Background technique [0002] Human papillomavirus (HPV) is a non-enveloped DNA virus with a diameter of about 50-60 nm. There are more than 100 types of HPV, and its infection can lead to various benign and malignant proliferative diseases of human skin and mucous membranes. High-risk HPV (type 16, 18, 45, 58) infection is closely related to the occurrence of malignant tumors such as cervical cancer. Low-risk HPV (type 6, type 11) infection causes venereal disease genital warts. The size of HPV genome is about 8kb, which is divided into non-coding region (NCR), early region (E region) and late region (L region). The noncoding region contains the origin of HPV DNA replication and regulator...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N15/65
Inventor 赵蔚明李靖刘娟王红唐伟孙允东周亚滨齐眉于晗
Owner SHANDONG UNIV
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