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Inhibin gene immunization on reproductive endocrinology and reproductive role

Author: ZuoDaGan
Tutor: YangLiGuo
School: Nanjing Agricultural College
Course: Animal Genetic Breeding and Reproduction
Keywords: Inhibin Genetic Immunity Follicular development Reproduction Endocrine
CLC: S852.4
Type: PhD thesis
Year: 2003
Downloads: 255
Quote: 5
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Abstract


In this paper, gene cloning, cell culture techniques, protein analysis techniques, such as enzyme immunoassay technology to build inhibin and hepatitis B surface antigen gene fusion expression plasmid, used to immunize animals to study the impact of the effect of inhibin gene immunization factors discussed inhibin gene immunization on reproductive endocrinology and reproductive role. Following major elements: a follistatin and hepatitis B surface antigen fusion gene expression plasmid and identification in order to build inhibin gene and hepatitis B surface antigen gene fusion eukaryotic expression plasmid, this study Anthwin software inhibin and hepatitis B surface antigen fusion gene structure and physicochemical properties of the product were analyzed to predict the antigenic epitopes. PCMV-S by PCR amplification of the S gene fragment and the restriction enzyme from the clone obtained plasmid pUI inhibin α (1-32) gene, then inhibin α (1-32) fusion gene fragment of S into the eukaryotic expression plasmid pcDNA3.1 (-); through digestion, sequencing is correct, using liposome-encapsulated the recombinant plasmids were transfected into HeLa cells to express, collect cells 3 days after transfection and G418 selection obtained positive clones, using SDS-PAGE and competition ELISA, to detect its expression product. The results showed that inhibin fusion gene product two structures, Helix accounted for 6%, Sheet accounted for 39%, Turn accounted for 8%, Coil 47%. Integrated hydrophilicity, antigenicity, hydrophobicity and other indicators, the first 225-258 amino acid sequences may contain epitopes of inhibin. For inhibin constructed recombinant eukaryotic expression plasmid by restriction endonuclease digestion and sequence analysis showed that the fusion gene fragment length 793bp, sequence and design exactly. Recombinant plasmids expressed in HeLa cells, the molecular weight of the fusion protein obtained 29KD. Fusion protein has immunological activity of inhibin, inhibin peptides with anti-inhibin antibodies compete for binding. These results show that the fusion constructed expression plasmid inhibin can expressed in eukaryotic cells, the recombinant protein having immunological activity of inhibin. 2 inhibin gene immunization immune response and its influencing factors in order to analyze the immune suppressing the immune response gene and its influencing factors, the tests were carried out two series of studies, the detection of antibodies inhibin P / N value and mouse antibodies ratio. Series one will inhibit the quality of the grain (pcINH) liposomal, sub 0μg (empty plasmid), 15μg, 25μg, 40μg four dose groups, intramuscular sexually mature rats interval of 20 days, with naked DNA to strengthen the immune once ( n = 10). Series two will inhibin fusion plasmid (pCIS) of 10μg, 50μg, 100μg three doses to empty plasmid and saline as control, intramuscular procaine hydrochloride treated by sexually mature rats, 20 days to strengthen the immune time interval (n = 18). For each group of 12 rats were immunized twice strengthening (n = 12), at intervals of 20 days. The results showed that one pcINH series by liposome-mediated immune rats twice receive a 50% (13/26) anti-inhibin antibody positive mice 20 days after the first immunization and 10 days after booster immunization, 40μg dose group The average inhibition antibody positive mice P / N value was higher than 15μg and 25μg antibody group P / N value, the increase in immunization dose did not increase the proportion of antibody-positive mice; Series 2 after immunization twice in pCIS procaine hydrochloride because treated rats, obtained 38.9% (23/54) of antibody-positive mice, obtained after the third immunization 55.6% (20/36) of antibody-positive mice, the dose can be increased to increase the immune antibody levels increased to strengthen the immune antibody positive mouse antibody positive rate and P / N value. But after the second booster immunization, 50μg and 100μg group reached the same proportion of antibody-positive mice. These results show that inhibin and hepatitis B surface antigen fusion gene expression plasmid immunized rats can produce anti-inhibin antibodies. Comprehensive analysis of the immunized mice against inhibin antibody levels and positive antibody levels in mice and found that the quality of grain pcINH inhibition by liposome-mediated immunity big dissertation: inhibin gene immunization on reproductive and reproductive endocrine function mouse, 40 \g dose of the highest levels of immune antibodies; inhibin immunization by fusion expression plasmid pCIS rats treated with procaine hydrochloride, 100 g doses similar to the levels of immunity produced by Hangzhou highest increase in the number of inhibin gene immunization can enhance inhibin the effect of genetic immunization, but immunization dose increases does not necessarily increase the percentage of antibody positive mice .3 inhibin gene immunization on reproductive and reproductive endocrinology To investigate the influence of inhibin gene immunization on reproductive endocrinology and reproductive role The two series of tests. series a used, 15,25,40 09 inhibin plasmid (PcINH) liposomal rats immunized 20 days, the same dose of naked DNA to strengthen the immune once daily (n = 10), follicular development was observed in rats (n two five). Department bad, J with two 10,50,100 \The rats treated with procaine hydrochloride (n = 18), 20 days after the booster immunization once follicular development rats (n = 6); the 12 rats in each group twice booster observe large rat follicular development (n = 6) and calving conditions (n ​​= 6). was found, PcINH immunized rats, 40 \immunized rats, 100 g dose group feeding twice and three times, respectively, the mature follicle immune count more than the control group, 6.9 and 7.5 Sand lt; 0 .05). pcIS twice after immunization, 100 \markedly increased Never lt; 0 .05). pcIS rats immunized three times the number of mature follicles was significantly increased after immunization than twice (35.2 ± 3.9 6.7 VS 31.0 soil, dead lt; 0.05). pCIs rats immunized three times after immunization group and the number of positive group placenta and litter size were more than the control group and negative group (p gt; 0.05). pe take H twice immune, pcls twice and three times after immunization positive immune mice Hangzhou body levels (P value added) and the number of mature ovarian follicles correlation coefficients were 0.13,0.43,0.45, pcls positive mice immunized three times Hang the levels associated with placental correlation coefficients of 0.77. Pc taken 10 days after the first immunization H FsH levels of positive and negative mice were significantly different between the mice do not lt; 0 .05), pcls immunized rats, and postpartum estrus blood loaded with higher levels of plasma FsH, Hangzhou body positive rat plasma FSH levels higher than the negative mice (P gt; 0.05). Pc Free State H twice remorse after plasma estradiol levels in rats was increased (p gt; 0

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CLC: > Agricultural Sciences > Livestock, animal medicine,hunting,silkworm,bee > Animal Medicine ( Veterinary Medicine) > Basic Veterinary Science > Animal Immunology
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