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Pichia pastoris of high copy expression recombination hepatitis B surface antigen, preparation and application thereof

A technology of hepatitis B surface antigen and yeast, which is applied in the field of constructs for transforming yeast cells, and can solve problems such as expensive blood sources, limited vaccine production and cost, and limited supply

Active Publication Date: 2008-12-03
SHANGHAI INST OF BIOLOGICAL PROD CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

One is to use blood-derived hepatitis B vaccine raw materials to prepare vaccines, but due to constraints such as expensive blood sources, limited supply, and difficulty in collection, the production and cost of this type of vaccine are greatly limited

Method used

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  • Pichia pastoris of high copy expression recombination hepatitis B surface antigen, preparation and application thereof
  • Pichia pastoris of high copy expression recombination hepatitis B surface antigen, preparation and application thereof
  • Pichia pastoris of high copy expression recombination hepatitis B surface antigen, preparation and application thereof

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preparation example Construction

[0069] The preparation method of the expression cassette of the present invention comprises the following steps:

[0070] (1) site-specific insertion of the HBsAg gene into a plasmid comprising the start signal element AOX and the stop signal element AOX (TT) to obtain a single-copy expression plasmid;

[0071] (2) Transforming the single-copy expression plasmid to obtain a complete HBsAg expression cassette.

[0072] In a preferred embodiment of the present invention, the plasmid used can be: pPIC3.5(K), pAO815 or pHIL-D2, etc., as long as it contains the start signal element AOX and the stop signal element AOX (TT); preferably pPIC3. 5(K) plasmid.

[0073] The HBsAg gene can be inserted between the 5'AOX1 and 3'AOX(TT) of the plasmid, preferably between the BamHI restriction site and the EcoR1 restriction site.

[0074] Conventional methods such as PCR can be used to amplify the HBsAg gene with the termination signal element 3'AOX (TT) from the constructed single-copy expr...

Embodiment 1

[0095] Example 1. Construction of Single Copy HBsAg Expression Plasmid

[0096] The HBsAg gene obtained from the hepatitis B virus genome was amplified by PCR method, inserted into the intracellular expression shuttle plasmid pPIC3.5(K) of Pichia pastoris, and constructed as a single-copy expression plasmid (see figure 1 ), and sequence the HBsAg gene.

Embodiment 2

[0097] Example 2. Transformation of a single-copy expression plasmid

[0098] Use the PCR method to amplify the HBsAg gene with the pPIC3.5(K) termination signal element 3'AOX(TT) from the constructed single-copy expression plasmid, and change the restriction sites at both ends, and the 5' end of the fragment is BglII site, and the 3' end is a BamHI site. Insert the fragment into the BamHI site of pPIC3.5 (K) to obtain a single-copy expression plasmid with a BamHI site at the 3' end of 3'AOX (TT) (see figure 2 ), and sequenced the HBsAg gene and its associated 3'AOX (TT).

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Abstract

The invention provides a yeast cell for expressing hepatitis B virus surface antigen. The yeast cells contain the constitution matter for expressing the hepatitis B virus surface antigen, and the constitution matter contain 5-15 tandem-arranged hepatitis B virus surface antigen expression cassettes. Each expression cassette includes the elements as follows: (a) an initial signal element AOX; (b) hepatitis B virus surface antigen genes and (c) a terminal signal element AOX (TT). The yeast cells are induced by methanol to express the hepatitis B virus surface antigen and form hepatitis B virus surface antigen virus particles with the expression amount being at least 30mg / L fermented solution. The invention also provides the constitution matter, the preparation method thereof, and a preparation method for the yeast cells and hepatitis B virus surface antigen virus particles expressed by the same. The constituted yeast cell with high copy number and high expression can be used for increasing the yield, reducing the cost, and is suitable for the large-scale production of the hepatitis B virus surface antigen vaccine.

Description

technical field [0001] The invention relates to the fields of bioengineering and immunity, in particular to a yeast cell expressing hepatitis B surface antigen, a construct for transforming the yeast cell and a construction method thereof. Background technique [0002] Hepatitis B is caused by hepatitis B virus (HBV), a serious worldwide disease that endangers human health. According to statistics, there are about 350 million chronic carriers of HBV in the world. About 1 million people die each year from diseases caused by HBV. One out of every 10 people in our country carries hepatitis B surface antigen (HBsAg). At present, there are about 120 million people in the country who are carriers of hepatitis B virus, and 28 million people are currently suffering from hepatitis B, with a prevalence rate of 2770 / 100,000. More than half of the population in China suffers from HBV infection, accounting for 80% of hepatitis worldwide. And one-third of people will develop into chron...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12N15/12C12N15/51C12P21/02
Inventor 何成楼觉人
Owner SHANGHAI INST OF BIOLOGICAL PROD CO LTD
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