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Method for preparing coagulation factor VIII by using rabbit mammary gland bioreactor

A bioreactor, coagulation factor technology, applied in biochemical equipment and methods, botanical equipment and methods, plant genetic improvement, etc., can solve problems such as increasing the risk of virus infection in patients

Inactive Publication Date: 2014-02-05
LANNUO BIOTECH WUXI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The raw materials of blood products generally come from donors. Due to limited testing technology, these blood products may carry infectious viruses, such as hepatitis B virus and immunodeficiency virus (HIV), which increases the risk of virus infection for patients

Method used

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  • Method for preparing coagulation factor VIII by using rabbit mammary gland bioreactor
  • Method for preparing coagulation factor VIII by using rabbit mammary gland bioreactor
  • Method for preparing coagulation factor VIII by using rabbit mammary gland bioreactor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1 Preparation of B-domain deleted recombinant human coagulation factor eighth cDNA (B-deleted rhFVIII cDNA)

[0028] Recombinant human full sequence coagulation factor VIII cDNA (rhFVIII cDNA), including A1, A2, B, A3, C1 and C2 domains, figure 1 It is a schematic diagram of the structure of B-deleted rhFVIII cDNA connected with bGHployA, "S" in the figure indicates the signal gene bGHployA, and B-deleted rhFVIII cDNA is artificially synthesized. The primers used to detect B-deleted rhFVIII cDNA by PCR detection method are pBClhF8-fs, pBClhF8-Ar, pBClhF8-CF, pBClhF8-r, pBClhF8-fA, figure 1 The direction indicated by the middle arrow is the direction of the primer.

[0029] Infect the B-deleted rhFVIII cDNA fragment into the competent cells of Zero Blunt TOPO bacteria (source: Invitrogen, Cat: K2800-20), the size of the B-deleted rhFVIII cDNA fragment is 4355bp, and then make the bacteria proliferate and extract the plasmid for Electrophoretic determination, th...

Embodiment 2

[0030] Example 2 Preparation of the Mammary Gland Expression Component pBCl-rhFVIII of the Eighth Coagulation Factor

[0031] Such as image 3 As shown, the pBCl plasmid is connected with chicken β-globulin insulator, goat β-casein promoter, TATA box, β-casein exon 1, β-casein intron 1, β-casein exon 2, Insert the B-deleted rhFVIII cDNA of the external signal gene into the restriction endonuclease Xho I multiple cloning site of the plasmid vector, and then connect -casein exons 7, 8, 9, β-casein introns 7, 8 , loading the β-casein genome fragment at the 3' end to form the mammary gland expression component pBCl-rhFVIII of the eighth blood coagulation factor, and the gene sequence is 25989bp long. The pBCl plasmid DNA contains the bacterial (EolEl) initiation fragment, the resistance gene AmpR promoter and the resistance gene AmpR.

[0032]Among them, chicken beta-globin insulator (Chicken beta-globin insulator), goat beta-casein promoter (Goat beta-casein promoter), TATA box...

Embodiment 3

[0033] Example 3 Amplification and detection of the mammary gland expression component pBCl-rhFVIII of the eighth coagulation factor

[0034] The mammary gland expression component pBCl-rhFVm of the eighth blood coagulation factor was transferred into the competent cells of the recipient strain DH5α, and the obtained recipient strain was multiplied to realize the amplification of pBCl-rhFVIII, and positive clones were screened out by PCR method, The primers are pBClhF8S-SNf: 5′-TGTGGTGAACTTCTCTAGACCCACCGTT-3′, pbCl-sr: 5′-CATCAGAAGTTAACAGCACAGTTAG-3′, and the PCR fragment size is 197bp. Then electrophoresis was carried out, and the results were as follows: Figure 4 As shown, among the 120 clones screened, numbers 38 and 47 are positive clones, wherein M is 100 bp DNA ladder (source: NEB, Cat. N0467S). Clones #38 and 47 were subjected to DNA sequencing, and clone #47 was completely consistent with the sequence of the gene bank cDNA (NM000132.3). The comparison results are as ...

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Abstract

The present invention discloses a method for preparing coagulation factor VIII by using a rabbit mammary gland bioreactor. The method comprises: inserting cDNA of B-domain deleted recombinant human coagulation factor VIII externally connected with a signal peptide into a plasmid vector to form a coagulation factor VIII mammary gland expression component, and performing microinjection or a somatic cell transplantation technology to obtain transgenic rabbit, wherein the present generation or future generation female rabbit of the obtained transgenic rabbit produces the recombinant human coagulation factor VIII in breast milk at the lactation period. According to the present invention, expression of the coagulation factor VIII mammary gland expression component in the transgenic rabbit mammary gland bioreactor can be achieved so as to prepare rhFVIII, and the transgenic rabbit is subjected to rapid population breeding so as to secrete a large amount of rhFVIII-containing breast milk, achieve batch production of the rhFVIII, reduce production cost to a great degree, avoid risk of virus infection on hFVIII extracted from blood plasma, and increase medication safety.

Description

technical field [0001] The invention relates to the field of preparation of recombinant protein drugs by non-human mammals, in particular to a method for preparing the eighth blood coagulation factor using a rabbit mammary gland bioreactor. Background technique [0002] Hemophilia is the most common bleeding disorder in humans. It is lifelong, recurrent and fatal. About one in every 5000-10000 men is a hemophiliac. Hemophilia is caused by the absence of certain functional clotting factors in the blood, such as the coenzyme coagulation factor VIII (in hemophilia A), and factor IX (in hemophilia B). Because hemophilia is caused by the deletion of a single X-linked gene encoding a circulating plasma protein, treatment of hemophilia focuses on the synthesis or extraction of normally functioning coagulation factors to replace the faulty or missing protein. [0003] The treatment of hemophilia is initially with whole plasma, followed by a concentrated preparation of highly purifi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/873
Inventor 薛非杨澜安礼友成勇杜福良
Owner LANNUO BIOTECH WUXI
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