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Human papilloma virus (HPV) resistant trivalent vaccine as well as preparation method and application thereof

A human papillomavirus, virus-like technology, applied in the fields of genetic engineering and vaccinology, can solve problems such as lack of low cost

Active Publication Date: 2014-03-26
TONGJI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] In short, there is still a lack of low-cost, high-efficiency methods for preparing various HPV L1 proteins or VLPs in this field, so there is an urgent need to develop new methods for preparing HPV L1 proteins or VLPs with high efficiency, simplicity, and low cost, especially for the preparation of HPV L1 proteins or VLPs. A method to simultaneously contain L1 proteins or VLPs of HPV16, HPV18, and HPV58 to enable the development of vaccines against multiple specific subtypes

Method used

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  • Human papilloma virus (HPV) resistant trivalent vaccine as well as preparation method and application thereof
  • Human papilloma virus (HPV) resistant trivalent vaccine as well as preparation method and application thereof
  • Human papilloma virus (HPV) resistant trivalent vaccine as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0184] Construction of high-efficiency expression system of HPV16L1 Pichia pastoris

[0185] 1. Optimization of HPV16L1 gene

[0186] Optimized sequence: According to the amino acid sequence of natural HPV16L1, the following factors are considered to optimize the design: the codon frequency used by Pichia pastoris, the G+C content in the gene and the secondary structure of mRNA.

[0187] The optimized nucleotide sequence of this embodiment is shown in SEQ ID NO:3, and the amino acid sequence of the HPV16L1 protein encoded by the sequence is shown in SEQ ID NO:2.

[0188] The optimized gene (SEQ ID NO: 3) was artificially synthesized, and BamHI and EcoRI restriction sites were introduced at its 5' end and 3' end respectively; the control sequence 1 used the wild-type HPV16L1 gene (SEQ ID NO: 1), Encoding the same HPV16L1 protein, after comparison, the homology between the optimized nucleic acid sequence and the control wild-type gene sequence is about 77.0%

[0189] 2. Kozak...

Embodiment 2

[0201] Construction of High Expression System of HPV18L1 Pichia pastoris

[0202] 1. Optimization of the target gene

[0203] Optimized sequence: According to the amino acid sequence of natural HPV18L1, the following factors are considered to optimize the design: the codon frequency used by Pichia pastoris, the G+C content in the gene, and the secondary structure of mRNA.

[0204] The nucleotide sequence of the target gene optimized in this example is shown in SEQ ID NO: 6, and the amino acid sequence of the HPV18L1 protein encoded by this sequence is identical to the natural sequence (shown in SEQ ID NO: 5). The optimized gene (SEQ ID NO: 6) was artificially synthesized, and BamHI and EcoRI restriction sites were introduced at its 5' end and 3' end respectively; the control sequence was the wild-type HPV18L1 gene (SEQ ID NO: 4), encoding For the same HPV18L1 protein, after comparison, the homology between the optimized sequence and the wild-type gene of the control is about...

Embodiment 3

[0211] Construction of High Expression System of HPV58L1 Pichia pastoris

[0212] Optimized sequence: According to the amino acid sequence of natural HPV58L1, the following factors are considered for optimal design: codon frequency used by Pichia pastoris, G+C content in gene, and secondary structure of mRNA.

[0213] The optimized nucleotide sequence of this embodiment is shown in SEQ ID NO:9, and the amino acid sequence of the gene encoding HPV58L1 protein is shown in SEQ ID NO:8. The optimized gene (SEQ ID NO:9) was artificially synthesized, and BamHI and EcoRI restriction sites were introduced at its 5' end and 3' end, respectively.

[0214] Control sequence 1: wild-type HPV58L1 gene (SEQ ID NO: 7), encoding the same HPV58L1 protein; after comparison, the homology between the optimized sequence and the control wild-type gene is about 77.2%.

[0215] 2. Kozak sequence optimization

[0216] The optimized kozak sequence 1 screened in Example 1 was selected, and the identifi...

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Abstract

The invention relates to a human papilloma virus (HPV) resistant trivalent vaccine as well as a preparation method and application thereof. Concretely, a kozak sequence capable of greatly improving the HPVL1 protein expression level is found through screening and analyzing a great number of kozak sequences, and genes 16, 18 and 58 of a main capsid protein L1 (HPV L1) of the HPV are redesigned, the sequences of the genes 16, 18 and 58 are optimized, a secondary structure of an mRNA (Messenger Ribonucleic Acid) causing influence to translation is eliminated, and then, a high-expression HPV L1 gene expression cassette is constructed on the basis. The expression cassette is particularly suitable for the expression of pichia pastoris and has the characteristics of high protein expression quantity and good stability.

Description

technical field [0001] The invention relates to the fields of genetic engineering and vaccinology, in particular, the invention relates to a trivalent vaccine against human papillomavirus and its preparation method and application. Background technique [0002] Cervical cancer is a common gynecological malignancy. Among the most common cancers in women, the incidence of cervical cancer ranks second. HPV (papillomavirus) virus infection is closely related to the occurrence of cervical cancer. At least 118 kinds of papillomaviruses have been isolated from humans so far. It has been confirmed that among anal and reproductive system cancers, cancers induced by HPV infection account for 3.7%. [0003] Among the HPV virus subtypes that have been isolated, 15 have been found to induce the occurrence of reproductive system cancer, so they are classified as high-risk HPV virus subtypes (such as HPV16, 18, 31, 33, 52 and 58), Two subtypes of HPV16 and HPV18 account for 70% of all ce...

Claims

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Application Information

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IPC IPC(8): C12N15/37C12N15/63C12N1/21C12N1/19C12N5/10C12P21/02C12N7/04A61K39/12A61P31/20C12R1/84
Inventor 潘卫庆徐新东张远斌
Owner TONGJI UNIV
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