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Method for synthesizing secreted human serum albumin employing bombyx mori posterior silk gland

A technology of human serum albumin and posterior silk gland, which is applied in the field of silkworm synthesis and secretion of exogenous proteins, can solve the problems of frequent allergic reactions and inability to purify products multiple times, so as to ensure biological safety, improve production efficiency and economical Benefits, the effect of reducing production costs

Inactive Publication Date: 2015-05-06
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the main source of human serum albumin is still separated and purified from human blood. However, there are often patients with blood-borne diseases such as hepatitis B, hepatitis C, and acquired immunodeficiency syndrome (AIDS) among blood donors. It will inevitably bring some pathogenic substances into the final product, and the current domestic technical means cannot purify the product multiple times. The purity of commercially available albumin is 95-97%, resulting in frequent allergic reactions after intravenous infusion

Method used

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  • Method for synthesizing secreted human serum albumin employing bombyx mori posterior silk gland
  • Method for synthesizing secreted human serum albumin employing bombyx mori posterior silk gland
  • Method for synthesizing secreted human serum albumin employing bombyx mori posterior silk gland

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Experimental program
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Embodiment 1

[0026] A) The pBHSA plasmid construction method and steps are as follows:

[0027] According to the human serum albumin (Human serum albumin, HSA) gene sequence, design the upstream primer DDDDK-HAS-F that contains enterokinase restriction site to amplify human serum albumin, as shown in SEQ ID NO.17: 5'- gatgatgatgataaggatgctcacaagagtgaggt-3'; the downstream primer HAS-R is shown in SEQ ID NO.18: 5'-ttagagacctaaggcagcttgactt-3', the HSA gene is amplified by PCR technique. The HSA PCR reaction system is shown in Table 1, and the HSA PCR reaction conditions are shown in Table 2.

[0028] Table 1 HSA PCR reaction system

[0029] name

Element

PrimeSTAR HS Polymerase

0.25ul

5×PrimeSTAR Buffer

2.5ul

dNTP Mixture

2ul

Upstream primer DDDDK-HSA-F

1ul

downstream primer HSA-R

1ul

HSA plasmid

0.1ul

wxya 2 o

up to 25

[0030] Table 2 HSA PCR reaction conditions

[0031]

[0032]The PC...

Embodiment 2

[0045] A) The pBHSA plasmid construction method and steps are as in Example 1.

[0046] B) pBHSA plasmid ( figure 1 ) and a helper plasmid capable of providing piggyBac transposase ( figure 2 ) were mixed at a ratio of 1:1, the total concentration of the two plasmids was 0.5 μg / μl, the plasmids were dissolved in 0.5 mM phosphate buffer solution containing 5 mM sodium chloride at pH=7, and then introduced into silkworms to lay eggs by microinjection Into fertilized eggs within 5 hours afterward, the total volume of introduction is 5nl. The microinjected silkworm eggs were reared to adults at 25° C., 80% humidity, and 12 hours of light, and crossed with non-transgenic silkworms (G1 generation). During the turning green stage of the G1 generation eggs in the transgenic experiment, a transgenic silkworm moth expressing the DsRed marker gene was observed by a fluorescence microscope (Olympus, SZX12, Japan), containing 15 transgenic positive silkworms, and the silkworms were bred...

Embodiment 3

[0053] A) The pBHSA plasmid construction method and steps are as in Example 1.

[0054] B) pBHSA plasmid ( figure 1 ) and a helper plasmid capable of providing piggyBac transposase ( figure 2 ) were mixed at a ratio of 1:1, the total concentration of the two plasmids was 0.45 μg / μl, the plasmids were dissolved in 0.6 mM phosphate buffer solution containing 4 mM sodium chloride at pH=7, and then introduced into silkworms to lay eggs by microinjection Within 4 hours after fertilization, the total volume of introduction is 12nl. The microinjected silkworm eggs were reared to adults at 25° C., 85% humidity, and 12 hours of light, and mated with non-transgenic silkworms for subsequent generation (G1 generation). During the turning green stage of the G1 generation eggs in the transgenic experiment, one transgenic silkworm moth expressing the DsRed marker gene was observed by a fluorescence microscope (Olympus, SZX12, Japan), and two transgenic positive silkworms were obtained. T...

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Abstract

The invention discloses a method for synthesizing secreted human serum albumin employing a bombyx mori posterior silk gland. The method comprises the following steps: firstly, constructing carrier pBHSA plasmids of human serum albumin secreted from bombyx mori, introducing the plasmids and auxiliary plasmids into bombyx mori fertilized ovum by virtue of micro-injection, introducing a red fluorescent protein gene and a human serum albumin gene into a bombyx mori genome by virtue of piggyBac transposons, and carrying out stable inheritance and expression to prepare transgenic bombyx mori; selfing, and carrying out homozygosis on the human serum albumin gene; and breeding the transgenic bombyx mori of secreting the human serum albumin. The transgenic bombyx mori is screened by virtue of a fluorescent marker gene; the secreted human serum albumin is specifically secreted by virtue of bombyx mori posterior silk gland cells; the human serum albumin production process is improved; the purification method is simplified; the production cost is reduced; a foundation is laid for improvement of the production efficiency of the human serum albumin and reduction of the production cost; and the biosecurity of human serum albumin products is ensured.

Description

technical field [0001] The invention relates to a method for synthesizing and secreting exogenous protein by silkworm, in particular to a method for synthesizing and secreting human serum albumin by using the rear silk gland of silkworm using transgenic technology. Background technique [0002] The mature human serum albumin molecule is composed of 585 amino acid residues and has a molecular weight of 66.5KDa. It is a soluble non-glycosylated globulin. Human serum albumin exists in human serum, accounting for more than 50% of the total serum protein, and has the functions of maintaining the colloid osmotic pressure of human blood and transporting metabolites and chemical drugs. Human serum albumin is an important biological product in modern medicine. It is used as a blood volume expander to supplement human protein and maintain normal blood circulation. For shock caused by blood loss, trauma and burns, cerebral edema and brain damage caused by elevated brain pressure, it h...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N15/14A01K67/04C07K14/765
Inventor 钟伯雄钱秋杰叶露鹏尤征英车家倩王少华宋佳张玉玉
Owner ZHEJIANG UNIV
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