Person-seminal-fluid cryoprotectant containing recombination person PRDX4 protein

A cryoprotectant and cryoprotection technology, which can be used in the preservation, application, and animal husbandry of human or animal bodies, and can solve the problems of increased DNA damage in sperm

Active Publication Date: 2016-01-06
刘娟 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Furthermore, Prdx4 knockout mice showed test

Method used

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  • Person-seminal-fluid cryoprotectant containing recombination person PRDX4 protein

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Experimental program
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Effect test

Embodiment 1

[0011] Embodiment 1: Preparation of recombinant human PRDX4 protein

[0012] Design and synthesize a pair of specific primers (upstream primer: 5'-tatccatatgtgggagacagaggagag-3', downstream primer: 5'-ttactcgagattcagtttatcgaaatac-3') according to the sequence encoding the human PRDX4 gene, and add restriction enzymes to the 5' ends of the upstream and downstream primers Sites NdeI and XhoI. The human epididymis mRNA extracted by conventional methods was subjected to RT-PCR to obtain cDNA, and then the human PRDX4 gene was amplified using the cDNA as a template. After double digestion with NdeI and XhoI, the amplified human PRDX4 gene fragment was inserted between the NdeI and XhoI restriction sites of the prokaryotic expression vector pET32b (+), and the prokaryotic expression vector pET32b (+) expressing human PRDX4 protein was obtained. ) / PRDX4. Then the vector is transformed into the expression strain OrigamiB (DE3), and the Escherichia coli genetically engineered strain ...

Embodiment 2

[0013] Embodiment 2: Preparation of human semen cryoprotectant containing recombinant human PRDX4 protein

[0014] Accurately weigh 1.5g of glucose, 1.3g of trisodium citrate dihydrate, and 1.3g of glycine, add pure water to 65ml, after completely dissolving, add 15ml of glycerin, and mix well. Then add 20ml egg yolk (obtained from fresh eggs), and place the suspension in a 56°C water bath for 40 minutes, stirring frequently, to make human semen cryoprotectant base liquid, which is also GEYC cryoprotectant. To the cooled human semen cryoprotection base solution, add the recombinant human PRDX4 protein obtained in Example 1 at a final concentration of 0.1 mg / ml, mix well, and finally filter with a 0.45 μm sterile filter to obtain recombinant human PRDX4 protein. Human semen cryoprotectant, aseptically divided, stored at -70°C, and used within 3 months.

Embodiment 3

[0015] Embodiment 3: the human semen cryoprotectant containing recombinant human PRDX4 protein is used for the cryopreservation of human semen

[0016] The present invention selects 50 semen samples from the Reproductive Center of Yantai Yuhuangding Hospital. The semen parameters meet the normal semen standards stipulated by the World Health Organization (WHO, 2010). The age is 28 to 35 years old. normal offspring.

[0017] Semen cryopreservation and recovery procedure: Mix the completely liquefied semen and cryoprotectant at a volume ratio of 2:1, incubate at 30-35°C for 5 minutes, and put them into cryovials. Put it in a refrigerator at 4°C for 1 hour, then suspend it in a mixture of liquid nitrogen vapor and air (-80°C to -100°C) for 15 minutes, and then put it into liquid nitrogen. After cryopreservation for 1 month, resuscitate in a 37°C water bath for 5-10 minutes.

[0018] After each portion of fresh semen is liquefied, it is divided into 3 portions and processed as f...

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Abstract

The invention provides a person-seminal-fluid cryoprotectant containing recombination person PRDX4 protein. The person-seminal-fluid cryoprotectant is prepared from the recombination person PRDX4 protein and person-seminal-fluid cryoprotective base liquid, wherein the adding amount of the recombination person PRDX4 protein in per 100 ml of person-seminal-fluid cryoprotective base liquid is 5 mg to 20 mg. Per 100 ml of person-seminal-fluid cryoprotective base liquid comprises 1.5 g of glucose, 1.3 g of trisodium citrate dehydrate, 1.3 g of glycine, 15 ml of glycerin, 20 ml of yolk and the balance purified water. According to the person-seminal-fluid cryoprotectant, the recombination person PRDX4 protein is used for cryopreservation of person seminal fluid for the first time, the activity, the survival rate and the sperm penetration rate of thawed person sperms are all remarkably higher than the activity, the survival rate and the sperm penetration rate of a control group (the person-seminal-fluid cryoprotective base liquid without the recombination person PRDX4 protein being added is used), the DNA damage rate is lower than that of the control group, and therefore it is proved that adding of the recombination person PRDX4 protein can improve the cryopreservation effect of the person-seminal-fluid cryoprotective base liquid on the person sperms.

Description

technical field [0001] The invention belongs to the field of cryoprotectants for human semen. Background technique [0002] According to the 2012 "China Infertility Status Survey Report", the incidence of infertility in China has increased from 3% 20 years ago to 15%, of which male factors account for 30% to 40%. Male infertility has become a A serious social problem. At present, assisted reproductive technology is an important means of treatment of male infertility, and semen cryopreservation is an indispensable part of it. Maternity insurance is provided for cancer patients undergoing radiotherapy and chemotherapy or couples who require fertility preservation. [0003] During the freezing and thawing process of semen, a large number of active oxygen free radicals are abnormally produced, and the process of freezing and thawing destroys the activity of antioxidant enzymes in semen. While the production is greatly increased, the oxidative stress scavenging ability natura...

Claims

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Application Information

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IPC IPC(8): A01N1/02
Inventor 刘娟朱鹏
Owner 刘娟
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