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Pig vesicula seminalis glandulae bioreactor and method for preparing recombinant protein or polypeptide with the same

A seminal vesicle and seminal plasma protein technology is applied in the field of transgenic pig seminal vesicle bioreactor, which can solve the problems of difficulty in collecting milk, difficult separation, low yield of exogenous protein, etc., and achieves mature collection and processing technology, advanced and simple methods, Simple effects of semen ingredients

Inactive Publication Date: 2008-10-29
YANGZHOU UNIV
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, in comparison, expression systems such as mammary glands also have some deficiencies that cannot be improved, such as a relatively long time interval from birth to first lactation; a long reproductive cycle; there is a longer lactation interval; some animals are more difficult to collect milk, Such as pigs, rabbits, etc.; in addition, some exogenous bioactive proteins expressed by mammary glands can be absorbed by animals, which may be harmful to animal health, especially when the expression level is relatively high
[0006] The disadvantage that the transgenic chicken bioreactor cannot overcome at present is that there are many differences in the embryonic development of poultry compared with mammals, and the research on it lags behind other animals for the time being, making the application of transgenic chickens still have many difficulties, resulting in Transgenic technology that has been widely used in animals is not suitable for application in poultry
For blood bioreactors, since the proteins in human and livestock blood are often similar, and the blood components are more complex, separation is more difficult; in addition, high concentrations of exogenous proteins in the blood may be harmful to the health of animals. Harmful, these unfavorable factors limit the application of blood expression system
The main problem of the urine expression system is that the yield of exogenous protein that can be expressed in urine is relatively low

Method used

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  • Pig vesicula seminalis glandulae bioreactor and method for preparing recombinant protein or polypeptide with the same
  • Pig vesicula seminalis glandulae bioreactor and method for preparing recombinant protein or polypeptide with the same
  • Pig vesicula seminalis glandulae bioreactor and method for preparing recombinant protein or polypeptide with the same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Embodiment 1 constructs gene expression vector

[0037] In order to construct a special expression vector that can be expressed in the boar seminal vesicles and secreted into the semen, this embodiment selects pcDNA3.0 as the basic framework for vector construction, and inserts various genetic elements into the transformed pcDNA3.0 vector in sequence.

[0038] 1. Transformation of pcDNA3.0: According to the analysis results of the restriction enzyme map, the pcDNA3.0 vector was digested with restriction enzyme Pvu II, the neo gene and its expression regulation sequence were excised, the digested products were separated by agarose gel electrophoresis, and DNA coagulation The 3.2kb fragment was recovered by the gel recovery kit, transformed into DH5α competent Escherichia coli after self-ligation, and a modified plasmid that did not contain the neo gene and its expression regulatory sequence was obtained from the positive transformed strain.

[0039] 2. Preparation and cl...

Embodiment 2

[0044] Example 2 Introduction of expression components into mouse genome by transgenic technology

[0045]The pcPSPII-EGFP expression vector was digested with NruI and XhoI, and after electrophoresis, the gel was cut to recover and purify the protein containing porcine seminal plasma protein II gene promoter, signal peptide sequence, coding sequence of green fluorescent protein reporter gene, and porcine seminal plasma protein II gene. The polyadenylation signal sequence and the target fragment of the 3' flanking region ( Figure 7 ). 30 micrograms of this fragment were wrapped with polycationic PEI and injected into the testes of six-week-old male mice for a total of three injections with an interval of 3 days. Counting from the last injection, 20 days later, the injected male mice were compared with normal female mice. Rats mate. After the hybrid mice were born, they were identified by PCR, and the transgenic positive mice were screened. And continue to breed transgenic p...

Embodiment 3

[0046] Example 3 Analysis of the expression of exogenous genes in transgenic male mice

[0047] The transgenic male mice of the F1 generation of the sexually mature transgenic positive male mice in Example 2 were cut open, and a piece of seminal vesicle gland tissue was excised respectively to make frozen sections. At the same time, a negative male mouse was taken to make a frozen section, and as a control, green fluorescence was observed under a fluorescent microscope, and green fluorescence was observed in the seminal vesicle gland tissue of the transgenic positive male mouse, while no green fluorescence was observed in the negative control male mouse ( Figure 9 ). Figure A is the tissue section of the seminal vesicles of the transgenic positive mice, and Figure B is the section of the seminal vesicles of the non-transgenic mice. After the transgenic positive male mice were mated with normal female mice, the vaginal suppository was collected, the vaginal suppository was di...

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Abstract

The invention discloses a boar seminal vesicle gland bioreactor and a method for producing recombinant protein or polypeptide. A gene expression vector specifically expressed in seminal vesicle gland of boar contains regulating elements such as promoter, signal peptide and DNA sequence of polyadenylation signal. When the expression vector is integrated with boar genome by transgenic technology, the recombinant protein or polypeptide can be specifically expressed in seminal vesicle gland tissue of adult boar and secreted in semen. The method can directly obtain recombinant protein or polypeptide from semen, and has the advantages of advanced and simple method and low cost. Compared with methods using other animals, the method has remarkable superiority in maintaining cost, production period, etc.

Description

technical field [0001] The present invention relates to a method for synthesizing recombinant proteins or polypeptides in transgenic pig seminal vesicles and secreting them into boar semen, more specifically, to a transgenic pig seminal vesicle bioreactor and the use of the bioreactor to produce recombinant proteins or peptide methods. Background technique [0002] At present, many precious medicinal proteins can be expressed in bacteria, fungi and yeast by DNA recombination technology, and produced through large-scale cultivation. However, the biological activity of many proteins is only possessed after post-processing, such as glycosylation, hydroxylation and carboxylation. In many cases, prokaryotic bacteria lack post-translational processing and modification capabilities, while yeast cells process expressed proteins differently from human proteins, and expressed products may be immunogenic or inactive. Although animal cell culture genetic engineering pharmaceutical sys...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N15/09C07K1/14
Inventor 宋成义高波王宵燕陈国宏赵文明滕尚辉谢飞王志跃
Owner YANGZHOU UNIV
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