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Human papilloma virus 58E7 protein expression and purification method and application

A human papillomavirus, 58E7 technology, applied in the biological field, can solve the problems of limited detection methods, false negatives, false positives, etc.

Inactive Publication Date: 2016-06-08
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] HPV58 is one of the main causes of cervical cancer. The current detection methods are very limited, such as PCR, which is easy to cause false positives or false negatives.

Method used

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  • Human papilloma virus 58E7 protein expression and purification method and application
  • Human papilloma virus 58E7 protein expression and purification method and application
  • Human papilloma virus 58E7 protein expression and purification method and application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Embodiment 1: Construction of HPV58 type E7 gene prokaryotic expression vector

[0027] The HPV-58 gene (SEQ ID NO.5) was amplified from HPV-58 positive human cervical epithelial cells by PCR method, and the primers HPV-58E7_Pup (SEQ ID NO.1) and HPV-58E7_Pdn (SEQ ID NO.2) used were synthesized by Qingke Biotechnology . The upstream and downstream primers for prokaryotic expression were respectively introduced into BamHI and EcoRI restriction sites

[0028] HPV-58E7_Pup: 5'-CCG GGATCC ATGAGAGGAAACAACCCAACG-3'

[0029] HPV-58E7_Pdn: 5'-CCG GAATTC TTATTGCTGTGCACAGCTAGG-3'

[0030] PCR reaction conditions: 5 μL of HPV-58 positive human cervical epithelial cell solution was added to 50 μL reaction system, 1 μL of 20 μmol / L upstream and downstream primers, 4 μL of 2.5 mmol / L dNTP mixture, 5 × Buffer (Mg 2+ plus) 10 μL, rTaq polymerase 0.5 μL, the rest with ddH 2 O make up. Using ABI company's (model 2720) PCR instrument, the PCR reaction conditions were pre-denaturatio...

Embodiment 2

[0032] Embodiment 2: the construction of HPV58 type E7 gene eukaryotic expression vector

[0033] The HPV-58 gene was amplified from the pGEX-4T2-(HPV-58E7) plasmid by PCR method, and the primers HPV-58E7_Eup (SEQ ID NO.3) and HPV-58E7_Edn (SEQ ID NO.4) used were synthesized by Qingke Bio. The upstream and downstream primers for eukaryotic expression were respectively introduced into EcoRI and BamHI restriction sites.

[0034] HPV-58E7_Eup: 5'-CCG GAATTC ATGAGAGGAAACAACCCAACGC-3'

[0035] HPV-58E7_Edn:'-CCG GGATCC TTATTGCTGTGCACAGCTAGG-3'

[0036] PCR reaction conditions: 1 μL of pGEX-4T2-(HPV-58E7) plasmid was added to 50 μL reaction system, 1 μL of 20 μmol / L upstream and downstream primers, 4 μL of 2.5 mmol / L dNTP mixture, 5×Buffer (Mg 2+ plus) 10 μL, rTaq polymerase 0.5 μL, the rest with ddH 2 O make up. Using ABI company's (model 2720) PCR instrument, the PCR reaction conditions were pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s; annealing at 60°...

Embodiment 3

[0038] Embodiment 3: Induced expression of HPV58 type E7 protein

[0039] The pGEX-4T2-(HPV-58E7) plasmid was transformed into Escherichia coli DH5α, inoculated in LB culture medium containing 100 μg / mL ampicillin (Amp), and when the OD value of the bacteria was between 0.6-0.8, IPTG was added to the final concentration 0.2mM, induction temperature: 26-28°C, collect bacteria after induction for 4-6h, lyse the bacteria by ultrasonic in ice bath, centrifuge at 12000rpm10min at 4°C to separate supernatant and precipitate, and identify by 15% polypropylene gel electrophoresis (see image 3 ).

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Abstract

The invention provides a human papilloma virus (HPV) 58E7 protein expression and purification method. The method includes: designing an amplification primer of the HPV-58E7, and effectively amplifying genes from HPV-58 positive cells; inserting a HPV-58E7 gene sequence containing different enzyme cutting sites into vectors pGEX-4T2 and pEGFP-C1 according to a sequence to obtain a recombinant vector; combining the recombinant vector with Glutathione-Sepharose 4B magnetic beads, cutting a GST tag to obtain HPV-58E7 protein, and subjecting the HPV-58E7 to PBC dialysis overnight purification. Through the HPV-58E7 protein obtained by the method, blood serum can be obtained through immune animals, and polyclonal antibodies with high specificity and high potency ratio can be obtained through purification; the HPV-58E7 protein can be applied in preparing the polyclonal antibodies.

Description

technical field [0001] The invention belongs to the field of biological technology, and relates to the induced expression of viral proteins and the preparation of antibodies, in particular to the prokaryotic expression of human papillomavirus type 58 E7 protein and its application in the preparation of polyclonal antibodies. Background technique [0002] Cervical cancer is one of the most common tumors of the female reproductive system. The incidence of cervical cancer in my country ranks second in the world, and it shows a trend of increasing and getting younger year by year. Among young women (≤30 years old), the incidence of cervical cancer in young women (≤30 years old) 2%-3% growth rate. Studies have confirmed that the persistent infection of high-risk human papillomavirus (HumanPapillomavirus, HPV) is the main cause of cervical cancer and precancerous lesions. HPV58 is a common subtype of high-risk HPV viruses. Among cervical cancer patients in my country, the number o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/025C07K16/08C12N15/37C12N15/70C12N15/79
CPCC07K14/005C07K16/084C07K2319/23C12N15/70C12N15/79C12N2710/20022
Inventor 程浩郑俏丽姜少杰陈贤祯朱江金纳
Owner ZHEJIANG UNIV
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