The objective of the present study was to evaluate the effect of an immunogenic maltose-binding protein-gonadotropin releasing hormone （GnRH-6-MBP） using genetic engineering. The synthetic mammalian tandem repeated ...The objective of the present study was to evaluate the effect of an immunogenic maltose-binding protein-gonadotropin releasing hormone （GnRH-6-MBP） using genetic engineering. The synthetic mammalian tandem repeated GnRH hexamer gene was inserted into the expression plasmid pMAL-c2x. Recombinant GnRH-6-MBP protein was over- expressed in E.coli strain BL21. Amylose resin with affinity chromatograph was used to purify target protein. The reactiongenicity of fusion protein was identified by indirect enzyme linked immunosorbent assay （ELISA）, and the antigenicity and biological effects of GnRH-6-MBP were tested in mice. In the experiment, 20 male Kunming white mice of 20 d old were randomly divided into treatment and control group. Ten mice were immunized with 100 μg GnRH-6-MBP administered subcutaneously （s.c.） thrice at 2-week intervals with GnRH-6-MBP. Mice were sacrificed after 3 weeks following the booster injection, the testis was removed, weighed and measured, and the histological structure was observed. The reactiongenicity of fusion protein to GnRH antibody was much higher than the control. Active immunization against GnRH-6-MBP reduced remarkably （P 〈 0.01） the length and weight of the testis, and shortened the girth and width of the testis （P 〈 0.05）, and suppressed testicular spermatogenesis compared to the control mice. These results indicate that the recombinant GnRH-6-MBP acted as a strong immunogen and caused atrophy of the testis.展开更多
MicroRNA(miRNA) has vital regulatory effects on the proliferation, differentiation and secretion of ovarian granulosa cells, but the role of miR-99a-5p in goat ovarian granulosa cells(GCs) is unclear. Both miR-99a-5p ...MicroRNA(miRNA) has vital regulatory effects on the proliferation, differentiation and secretion of ovarian granulosa cells, but the role of miR-99a-5p in goat ovarian granulosa cells(GCs) is unclear. Both miR-99a-5p and Frizzled-5(FZD5) were found to be expressed in GCs in goat ovaries via fluorescence in situ hybridization and immunohistochemistry, respectively, and FZD5 was verified(P<0.001) as a target gene of miR-99a-5p by double luciferase reporter gene experiments. Furthermore, FZD5 mRNA and protein expression were both found to be regulated(P<0.05) by miR-99a-5p in GCs. Moreover, the overexpression of miR-99a-5p or knockdown of FZD5 suppressed(P<0.05) estradiol and progesterone secretion from the GCs, as determined by ELISA. In summary, miR-99a-5p inhibits target gene FZD5 expression and estradiol and progesterone synthesis in GCs. Our study thus provides seminal data and new insights into the regulatory mechanisms of follicular development in the goat and other animals.展开更多
Skeletal muscle accounts for about 40% of mammalian body weight, the development of which is a dynamic, complex and precisely regulated process that is critical for meat production. We here described the transcriptome...Skeletal muscle accounts for about 40% of mammalian body weight, the development of which is a dynamic, complex and precisely regulated process that is critical for meat production. We here described the transcriptome expression profile in 21 goat samples collected at 7 growth stages from fetus to kid, including fetal 45(F45), 65(F65), 90(F90), 120(F120), and 135(F135) days, and birth 1(B1) day and 90(B90) days kids. Paraffin sections combined with RNA-seq data of the 7 stages divided the transcriptomic functions of skeletal muscle into 4 states: before F90, F120, F135 and B1, and B90. And the dynamic expression of all 4 793 differentially expressed genes(DEGs) was identified. Furthermore, DEGs were clustered by weighted gene correlation network analysis into 4 modules(turquoise, grey, blue and brown) that corresponded to these 4 states. Functional and pathway analysis indicated that the active genes in the stages before F90(turquoise) were closely related to skeletal muscle proliferation. The DEGs in the F120-related module(grey) were found to participate in the regulation of skeletal muscle structure and skeletal muscle development by regulating t RNA. The brown module(F135 and B1) regulated fatty acid biological processes to maintain the normal development of muscle cells. The DEGs of B90 high correlation module(blue) were involved the strengthening and power of skeletal muscle through the regulation of actin filaments and tropomyosin. Our current data thus revealed the internal functional conversion of the goat skeletal muscle in the growth from fetus to kid. The results provided a theoretical basis for analyzing the involvement of m RNA in skeletal muscle development.展开更多
基金the support from the Natural Science Foundation of Anhui Provincial Education Department, China (KJ2007B175)
文摘The objective of the present study was to evaluate the effect of an immunogenic maltose-binding protein-gonadotropin releasing hormone （GnRH-6-MBP） using genetic engineering. The synthetic mammalian tandem repeated GnRH hexamer gene was inserted into the expression plasmid pMAL-c2x. Recombinant GnRH-6-MBP protein was over- expressed in E.coli strain BL21. Amylose resin with affinity chromatograph was used to purify target protein. The reactiongenicity of fusion protein was identified by indirect enzyme linked immunosorbent assay （ELISA）, and the antigenicity and biological effects of GnRH-6-MBP were tested in mice. In the experiment, 20 male Kunming white mice of 20 d old were randomly divided into treatment and control group. Ten mice were immunized with 100 μg GnRH-6-MBP administered subcutaneously （s.c.） thrice at 2-week intervals with GnRH-6-MBP. Mice were sacrificed after 3 weeks following the booster injection, the testis was removed, weighed and measured, and the histological structure was observed. The reactiongenicity of fusion protein to GnRH antibody was much higher than the control. Active immunization against GnRH-6-MBP reduced remarkably （P 〈 0.01） the length and weight of the testis, and shortened the girth and width of the testis （P 〈 0.05）, and suppressed testicular spermatogenesis compared to the control mice. These results indicate that the recombinant GnRH-6-MBP acted as a strong immunogen and caused atrophy of the testis.
基金supported by the National Natural Science Foundation of China(31772566 and 31972629)the Central Guidance on Local Science and Technology Development Fund of Anhui Province,China(202007d06020005)。
文摘MicroRNA(miRNA) has vital regulatory effects on the proliferation, differentiation and secretion of ovarian granulosa cells, but the role of miR-99a-5p in goat ovarian granulosa cells(GCs) is unclear. Both miR-99a-5p and Frizzled-5(FZD5) were found to be expressed in GCs in goat ovaries via fluorescence in situ hybridization and immunohistochemistry, respectively, and FZD5 was verified(P<0.001) as a target gene of miR-99a-5p by double luciferase reporter gene experiments. Furthermore, FZD5 mRNA and protein expression were both found to be regulated(P<0.05) by miR-99a-5p in GCs. Moreover, the overexpression of miR-99a-5p or knockdown of FZD5 suppressed(P<0.05) estradiol and progesterone secretion from the GCs, as determined by ELISA. In summary, miR-99a-5p inhibits target gene FZD5 expression and estradiol and progesterone synthesis in GCs. Our study thus provides seminal data and new insights into the regulatory mechanisms of follicular development in the goat and other animals.
基金supported by the National Natural Science Foundation of China (31772566 and 31972629)the Anhui Key Research and Development Program, China (1804a07020128)the Anhui Province Academic and Technology Leader Reserve Talent Project, China (2019H206)。
文摘Skeletal muscle accounts for about 40% of mammalian body weight, the development of which is a dynamic, complex and precisely regulated process that is critical for meat production. We here described the transcriptome expression profile in 21 goat samples collected at 7 growth stages from fetus to kid, including fetal 45(F45), 65(F65), 90(F90), 120(F120), and 135(F135) days, and birth 1(B1) day and 90(B90) days kids. Paraffin sections combined with RNA-seq data of the 7 stages divided the transcriptomic functions of skeletal muscle into 4 states: before F90, F120, F135 and B1, and B90. And the dynamic expression of all 4 793 differentially expressed genes(DEGs) was identified. Furthermore, DEGs were clustered by weighted gene correlation network analysis into 4 modules(turquoise, grey, blue and brown) that corresponded to these 4 states. Functional and pathway analysis indicated that the active genes in the stages before F90(turquoise) were closely related to skeletal muscle proliferation. The DEGs in the F120-related module(grey) were found to participate in the regulation of skeletal muscle structure and skeletal muscle development by regulating t RNA. The brown module(F135 and B1) regulated fatty acid biological processes to maintain the normal development of muscle cells. The DEGs of B90 high correlation module(blue) were involved the strengthening and power of skeletal muscle through the regulation of actin filaments and tropomyosin. Our current data thus revealed the internal functional conversion of the goat skeletal muscle in the growth from fetus to kid. The results provided a theoretical basis for analyzing the involvement of m RNA in skeletal muscle development.
