The Chinese tree shrew(Tupaia belangeri chinensis),a member of the mammalian order Scandentia,exhibits considerable similarities with primates,including humans,in aspects of its nervous,immune,and metabolic systems.Th...The Chinese tree shrew(Tupaia belangeri chinensis),a member of the mammalian order Scandentia,exhibits considerable similarities with primates,including humans,in aspects of its nervous,immune,and metabolic systems.These similarities have established the tree shrew as a promising experimental model for biomedical research on cancer,infectious diseases,metabolic disorders,and mental health conditions.Herein,we used metatranscriptomic sequencing to analyze plasma,as well as oral and anal swab samples,from 105 healthy asymptomatic tree shrews to identify the presence of potential zoonotic viruses.In total,eight mammalian viruses with complete genomes were identified,belonging to six viral families,including Flaviviridae,Hepeviridae,Parvovirinae,Picornaviridae,Sedoreoviridae,and Spinareoviridae.Notably,the presence of rotavirus was recorded in tree shrews for the first time.Three viruses-hepacivirus 1,parvovirus,and picornavirus-exhibited low genetic similarity(<70%)with previously reported viruses at the whole-genome scale,indicating novelty.Conversely,three other viruses-hepacivirus 2,hepatovirus A and hepevirus-exhibited high similarity(>94%)to known viral strains.Phylogenetic analyses also revealed that the rotavirus and mammalian orthoreovirus identified in this study may be novel reassortants.These findings provide insights into the diverse viral spectrum present in captive Chinese tree shrews,highlighting the necessity for further research into their potential for crossspecies transmission.展开更多
Tupaia belangeri,as a typical tropical species,has high value in biological research.It has the characteristics of small body size,high brain to body mass ratio,short reproductive cycle,short lifespan,and low maintena...Tupaia belangeri,as a typical tropical species,has high value in biological research.It has the characteristics of small body size,high brain to body mass ratio,short reproductive cycle,short lifespan,and low maintenance cost.In biomedical research,experimental animals considered as substitutes for primates.This review discusses the systematic classification,distribution,and research progress of Scandentia.Emphasis was focus on the research progress,developmental status,and basic biological research in T.belangeri.Believing that the T.belangeri,as the closest species to humans,has many advantages as an experimental animal,and it can be further widely applied in human disease research.Therefore,how to create a clear and stable strain of T.belangeri with a clear genetic background is crucial for the development of the national economy and scientific research.展开更多
鉴于原猿类的树鼩在进化中的特殊地位,有关它的神经生物学研究十分活跃。树鼩的单位放电研究已有报告(J. E. Abano et al. 1978;A. L. Humphrey et al. 1977、1980),但是采用慢性微电极技术记录行为状态的树鼩的单位放电方法迄今未见报...鉴于原猿类的树鼩在进化中的特殊地位,有关它的神经生物学研究十分活跃。树鼩的单位放电研究已有报告(J. E. Abano et al. 1978;A. L. Humphrey et al. 1977、1980),但是采用慢性微电极技术记录行为状态的树鼩的单位放电方法迄今未见报道,本文报告一种记录清醒活动状态树鼩的单位放电技术考虑到树鼩形体甚小、体重仅100多克。要分离单位放电,必须拥有一种特殊规格的微型微推进器,以适应特殊实验之需。本研究设计一种采用差动结构原理实现微推进的装置,整个装置由微推进器与基座组成。微推进器的参数如下:重量8.8克;微调范围:2.0毫米;微调读数5微米;微调可控范围:小于2微米;外形尺寸:13×13×51毫米。微推进器相对于基座有直径为1.8毫米的径向偏心移动范围。基座重0.65克,实验在局麻下,在立体定向仪控制下埋植基座。术后第二天即可实验观察。展开更多
根据“Aschoff rule”,昼行性动物近似昼夜节律周期的长度和光照强度呈负相关,活动时间与休息时间的比率以及活动量均与光照强度呈正相关。而夜行性动物的情况则相反(比宁,1964;Aschoff,1959,1960;Sollberger,1965)。这在爬行动物、鱼...根据“Aschoff rule”,昼行性动物近似昼夜节律周期的长度和光照强度呈负相关,活动时间与休息时间的比率以及活动量均与光照强度呈正相关。而夜行性动物的情况则相反(比宁,1964;Aschoff,1959,1960;Sollberger,1965)。这在爬行动物、鱼、鸟以及夜间活动的哺乳类已得到了肯定的证实,但对一些白天活动的哺乳类还不清楚。到目前为止,检查了7种白天活动的灵长类动物均不符合这条规律(Tokura et al.,1978,1979,1980)。展开更多
The tree shrew （Tupaia belangeri） is a promising laboratory animal that possesses a closer genetic relationship to primates than to rodents. In addition, advantages such as small size, easy breeding, and rapid repro...The tree shrew （Tupaia belangeri） is a promising laboratory animal that possesses a closer genetic relationship to primates than to rodents. In addition, advantages such as small size, easy breeding, and rapid reproduction make the tree shrew an ideal subject for the study of human disease. Numerous tree shrew disease models have been generated in biological and medical studies in recent years. Here we summarize current tree shrew disease models, including models of infectious diseases, cancers, depressive disorders, drug addiction, myopia, metabolic diseases, and immune-related diseases. With the success of tree shrew transgenic technology, this species will be increasingly used in biological and medical studies in the future.展开更多
AIM: To further analyze the interaction of tupaia CD81 with hepatitis C virus (HCV) envelope protein E2. METHODS: A tupaia CD81 large extracellular loop (CD81 LEL), which binds to HCV E2 protein, was cloned and expres...AIM: To further analyze the interaction of tupaia CD81 with hepatitis C virus (HCV) envelope protein E2. METHODS: A tupaia CD81 large extracellular loop (CD81 LEL), which binds to HCV E2 protein, was cloned and expressed as a GST-fusion protein, and interaction of HCV E2 protein with a tupaia CD81 LEL was evaluated by enzyme-linked immunosorbent assay (EIA). RESULTS: Although tupaia and human CD81 LEL differed in 6 amino acid changes, tupaia CD81 LEL was strongly recognized by anti-CD81 antibodies against human CD81 LEL conformation-dependent epitopes. Investigating LEL CD81-E2 interactions by EIA, we demonstrated that binding of tupaia CD81 LEL GST fusion protein to recombinant HCV E2 protein was markedly reduced compared to binding of human CD81 LEL GST fusion protein to recombinant HCV E2 protein. CONCLUSION: These data suggest that the structural differences in-between the tupaia and human CD81 may alter the interaction of the large extracellular loop with HCV envelope glycoprotein E2. These findings may be important for the understanding of the mechanisms of binding and entry of HCV to PTHs.展开更多
To characterize the patterns of ABR waves in tree shrews, we must understand the hearing sensitivity and auditory function of healthy adult tree shrews. Fifteen tree shrews(30 ears) were stimulated with clicks and ton...To characterize the patterns of ABR waves in tree shrews, we must understand the hearing sensitivity and auditory function of healthy adult tree shrews. Fifteen tree shrews(30 ears) were stimulated with clicks and tone-pips at 11 different frequencies from 1 to 60 kHz. The ABR waves were recorded and analyzed. The ABR consisted of five to seven positive waves in the first 10 ms after a click stimulus, and the average hearing threshold of component III was 27.86 ± 3.78 dB SPL. Wave III was the largest and most clear. The ABR threshold was related to the tone-pip sitmulus by a "U" shaped curve. The sensitive frequency was approximately 8 kHz in tree shrews. The latencies systematically decreased with increasing stimulus frequencies. The ABR amplitudes of wave III increased as the sound pressure level increased. All of these results provide an empirical basis for future studies of hearing diseases in tree shrews.展开更多
Objective:To investigate the ectoparasite communities on Tupaia belangeri (T. belangeri) in the surrounding areas of Erhai Lake and their potential importance in medical and veterinary science. Methods: Different loca...Objective:To investigate the ectoparasite communities on Tupaia belangeri (T. belangeri) in the surrounding areas of Erhai Lake and their potential importance in medical and veterinary science. Methods: Different locations in surrounding areas of Erhai Lake in Yunnan were selected as the investigation spots. T. belangeri were randomly captured with baited mouse cages and then brought to the laboratory for identification according to their body shape, size and color, and measurements such as the lengths of their body, ear and hind feet. Ectoparasites on them were collected and identified to determine the species under microscope after specimens were mounted on slides, with the clearing and drying process completed. The constituent ratio (C), prevalence (P) and average ectoparasite abundance (A) were used to test the prevalence and density of ectoparasites; Non-parametric Mann-Whitney U-tests were used to test the differences in the abundance and richness of ectoparasites between female and male hosts; Spearman correlation analysis was used to analyze the relationship between ectoparasites and the body parameters of hosts. Results: There was very high species diversity of ectoparasites found on T. belangeri and some species of ectoparasites were reported associated with human diseases. A high proportion (88%) of 107 T. belangeri was found to be infested with ectoparasites. A total of 75 species of ectoparasites on them were collected, including 58 species of chigger mite, 12 species of gamasid mite, 4 species of flea and 1 species of sucking louse. Within this ectoparasite complex, 13 species were previously reported to be vectors of human disease agents. Non-parametric Mann-Whitney U-tests showed significant differences between male and female T. belangeri in terms of the abundance and richness of total ectoparasites. However, the species richness and abundance of chigger mites, sucking lice, fleas or gamasid mites presented no significant differences between male and female hosts. Spearman correlation analysis showed that none of correlations were observed between host body parameters with abundance and richness of total ectoparasites, chigger mites, suckinglice, fleas or gamatid mites. Conclusion: The results suggest that there is very high species diversity of ectoparasites on T. belangeri, consisting mainly of communities of sucking lice, fleas, chiggers and gamasid mites.展开更多
Physiological adaptation of tree shrews(Tupaia belangeri)to changing environmental temperature has been re-ported in detail.However,the T.belangeri origin(mainland or island),population history,and adaptation to histo...Physiological adaptation of tree shrews(Tupaia belangeri)to changing environmental temperature has been re-ported in detail.However,the T.belangeri origin(mainland or island),population history,and adaptation to histor-ical climate change remain largely unknown or controversial.Here,for thefirst time,we sequenced the simplified genome of 134 T.belangeri individuals from 12 populations in China and further resequenced one individual from each population.Using population genomic approaches,wefirst observed considerable genetic variation in T.be-langeri.Moreover,T.belangeri populations formed obvious genetic structure and reflected different demographic histories;they generally exhibited high genetic diversity,although the isolated populations had relatively low ge-netic diversity.The results presented in this study indicate that T.b.modesta and T.b.tonquinia were separated recently and with a similar population dynamics.Second,physical barriers rather than distance were the driving factors of divergence,and environmental heterogeneity may play an important role in genetic differentiation in T.belangeri.Moreover,our analyses highlight the role of historical global climates in the T.belangeri population dynamics and indicate that the decrease of the T.belangeri population size may be due to the low temperature.Finally,we identified the olfaction-associated adaptive genes between different altitude populations and found that olfactory-related genes of high-altitude populations were selectively eliminated.Our study provides demographic history knowledge of T.belangeri;their adaption history offers new insights into their evolution and adaptation,and provides valuable baseline information for conservation measures.展开更多
目的对丙型病毒性肝炎（hepatitis C virus，HCV）小动物模型树嗣的干扰素刺激基因15（interferon stimulated gene15，ISG15）分子的全长eDNA序列进行克隆及分子生物学功能分析，为树晌模型在HCV感染天然免疫中的研究提供分子生物学基...目的对丙型病毒性肝炎（hepatitis C virus，HCV）小动物模型树嗣的干扰素刺激基因15（interferon stimulated gene15，ISG15）分子的全长eDNA序列进行克隆及分子生物学功能分析，为树晌模型在HCV感染天然免疫中的研究提供分子生物学基础。方法根据Genbank中灵长类及其他哺乳类动物ISG15分子序列保守区设计引物，使用SmarterRace方法扩增树购ISG15全长序列。在序列两端设计特异性引物，引入酶切位点，进行全长片段扩增。全长PCR产物纯化回收后连接至pMD18-T载体，构建重组质粒pMD18-T-tbISG15。对重组质粒进行酶切鉴定、测序。对序列进行同源性及种系进化，同时使用SWISSMODEL同源建模方法进行蛋白二级、三级结构预测及功能分析。结果获得树购ISG15全长序列共687bp，编码157个氨基酸。树晌ISG15与其他哺乳动物ISG15高度同源，与人的核苷酸及氨基酸序列同源性分别高达72．99％及71．34％，核苷酸及氨基酸序列进化树分析均显示树嗣种系最为接近灵长类动物。结构域分析提示：树嗣ISG15主要由两个类泛素样结构域构成。软件预测所得树购ISG15三维结构与人ISG15三维结构高度相似，且具有类泛素样三维结构，动力学检测提示本试验预测的树嗣ISG15三维结构稳定，可信度高。结论对树胸ISG15的克隆进一步完善了对树购模型的认识。为进一步在体内研究HCV感染的天然免疫机制奠定了分子生物学基础。展开更多
The human liver chimeric mouse is a milestone animal model for hepatitis B virus(HBV)infection.Such mice with primary human hepatocyte(PHH)transplantation are adequate to support chronic HBV infection for several week...The human liver chimeric mouse is a milestone animal model for hepatitis B virus(HBV)infection.Such mice with primary human hepatocyte(PHH)transplantation are adequate to support chronic HBV infection for several weeks and to evaluate antiviral drugs.However,the drawbacks of PHHs include lack of available donors,poor expansion in vitro and ethical issues that limit the application of human liver chimeric mice,necessitating the search for alternatives.Here,we transplanted primary tupaia hepatocytes(PTHs)into the livers of immunodeficient mice and achieved high liver chimerism within six weeks.These tupaia liver chimeric mice are adequate to support chronic infection of the four common HBV genotypes A,B,C and D for 36 weeks,as well as evaluate of antiviral drugs,including hepatitis B immune globulin(HBIG),monoclonal antibody and nucleoside analogues(NAs),for preventative therapy and treatment post infection.In conclusion,the tupaia liver chimeric mouse model provides a convenient,efficient and stable animal model for chronic HBV infection and long-term drug evaluation.展开更多
基金supported by the National Key R&D Program of China (2021YFC2300900,2021YFC2301300)Academic Promotion Programme of Shandong First Medical University (2019QL006)+2 种基金Natural Science Foundation of Shandong Province (ZR2020QH274)Yunnan Key Research and Development Program (202103AQ100001,202102AA310055)Key Program of Chinese Academy of Sciences (KJZD-SW-L11)。
文摘The Chinese tree shrew(Tupaia belangeri chinensis),a member of the mammalian order Scandentia,exhibits considerable similarities with primates,including humans,in aspects of its nervous,immune,and metabolic systems.These similarities have established the tree shrew as a promising experimental model for biomedical research on cancer,infectious diseases,metabolic disorders,and mental health conditions.Herein,we used metatranscriptomic sequencing to analyze plasma,as well as oral and anal swab samples,from 105 healthy asymptomatic tree shrews to identify the presence of potential zoonotic viruses.In total,eight mammalian viruses with complete genomes were identified,belonging to six viral families,including Flaviviridae,Hepeviridae,Parvovirinae,Picornaviridae,Sedoreoviridae,and Spinareoviridae.Notably,the presence of rotavirus was recorded in tree shrews for the first time.Three viruses-hepacivirus 1,parvovirus,and picornavirus-exhibited low genetic similarity(<70%)with previously reported viruses at the whole-genome scale,indicating novelty.Conversely,three other viruses-hepacivirus 2,hepatovirus A and hepevirus-exhibited high similarity(>94%)to known viral strains.Phylogenetic analyses also revealed that the rotavirus and mammalian orthoreovirus identified in this study may be novel reassortants.These findings provide insights into the diverse viral spectrum present in captive Chinese tree shrews,highlighting the necessity for further research into their potential for crossspecies transmission.
基金supported by the National Natural Science Foundation of China(32160254)Yunnan Ten Thousand Talents Plan Young&Elite Talents Project(YNWR-QNRC-2019-047)。
文摘Tupaia belangeri,as a typical tropical species,has high value in biological research.It has the characteristics of small body size,high brain to body mass ratio,short reproductive cycle,short lifespan,and low maintenance cost.In biomedical research,experimental animals considered as substitutes for primates.This review discusses the systematic classification,distribution,and research progress of Scandentia.Emphasis was focus on the research progress,developmental status,and basic biological research in T.belangeri.Believing that the T.belangeri,as the closest species to humans,has many advantages as an experimental animal,and it can be further widely applied in human disease research.Therefore,how to create a clear and stable strain of T.belangeri with a clear genetic background is crucial for the development of the national economy and scientific research.
文摘鉴于原猿类的树鼩在进化中的特殊地位,有关它的神经生物学研究十分活跃。树鼩的单位放电研究已有报告(J. E. Abano et al. 1978;A. L. Humphrey et al. 1977、1980),但是采用慢性微电极技术记录行为状态的树鼩的单位放电方法迄今未见报道,本文报告一种记录清醒活动状态树鼩的单位放电技术考虑到树鼩形体甚小、体重仅100多克。要分离单位放电,必须拥有一种特殊规格的微型微推进器,以适应特殊实验之需。本研究设计一种采用差动结构原理实现微推进的装置,整个装置由微推进器与基座组成。微推进器的参数如下:重量8.8克;微调范围:2.0毫米;微调读数5微米;微调可控范围:小于2微米;外形尺寸:13×13×51毫米。微推进器相对于基座有直径为1.8毫米的径向偏心移动范围。基座重0.65克,实验在局麻下,在立体定向仪控制下埋植基座。术后第二天即可实验观察。
文摘根据“Aschoff rule”,昼行性动物近似昼夜节律周期的长度和光照强度呈负相关,活动时间与休息时间的比率以及活动量均与光照强度呈正相关。而夜行性动物的情况则相反(比宁,1964;Aschoff,1959,1960;Sollberger,1965)。这在爬行动物、鱼、鸟以及夜间活动的哺乳类已得到了肯定的证实,但对一些白天活动的哺乳类还不清楚。到目前为止,检查了7种白天活动的灵长类动物均不符合这条规律(Tokura et al.,1978,1979,1980)。
基金supported by the National Nature Science Foundation of China(81325016,U1602221,81322038 and U1502222)
文摘The tree shrew （Tupaia belangeri） is a promising laboratory animal that possesses a closer genetic relationship to primates than to rodents. In addition, advantages such as small size, easy breeding, and rapid reproduction make the tree shrew an ideal subject for the study of human disease. Numerous tree shrew disease models have been generated in biological and medical studies in recent years. Here we summarize current tree shrew disease models, including models of infectious diseases, cancers, depressive disorders, drug addiction, myopia, metabolic diseases, and immune-related diseases. With the success of tree shrew transgenic technology, this species will be increasingly used in biological and medical studies in the future.
基金Supported by Grants from Health Department of Hubei Province (JX2B09)NSFC (30771911)National 973 Key Program (2009CB522502), and Tongji Hospital
文摘AIM: To further analyze the interaction of tupaia CD81 with hepatitis C virus (HCV) envelope protein E2. METHODS: A tupaia CD81 large extracellular loop (CD81 LEL), which binds to HCV E2 protein, was cloned and expressed as a GST-fusion protein, and interaction of HCV E2 protein with a tupaia CD81 LEL was evaluated by enzyme-linked immunosorbent assay (EIA). RESULTS: Although tupaia and human CD81 LEL differed in 6 amino acid changes, tupaia CD81 LEL was strongly recognized by anti-CD81 antibodies against human CD81 LEL conformation-dependent epitopes. Investigating LEL CD81-E2 interactions by EIA, we demonstrated that binding of tupaia CD81 LEL GST fusion protein to recombinant HCV E2 protein was markedly reduced compared to binding of human CD81 LEL GST fusion protein to recombinant HCV E2 protein. CONCLUSION: These data suggest that the structural differences in-between the tupaia and human CD81 may alter the interaction of the large extracellular loop with HCV envelope glycoprotein E2. These findings may be important for the understanding of the mechanisms of binding and entry of HCV to PTHs.
基金supported by Natural Science Foundation of China[grant numbers 81760188]Scientific Research Fund of Guangxi Provincial Education Department [grant numbers 2017KY0103 (02601217023C)]
文摘To characterize the patterns of ABR waves in tree shrews, we must understand the hearing sensitivity and auditory function of healthy adult tree shrews. Fifteen tree shrews(30 ears) were stimulated with clicks and tone-pips at 11 different frequencies from 1 to 60 kHz. The ABR waves were recorded and analyzed. The ABR consisted of five to seven positive waves in the first 10 ms after a click stimulus, and the average hearing threshold of component III was 27.86 ± 3.78 dB SPL. Wave III was the largest and most clear. The ABR threshold was related to the tone-pip sitmulus by a "U" shaped curve. The sensitive frequency was approximately 8 kHz in tree shrews. The latencies systematically decreased with increasing stimulus frequencies. The ABR amplitudes of wave III increased as the sound pressure level increased. All of these results provide an empirical basis for future studies of hearing diseases in tree shrews.
基金Supported by the Natural Science Foundation of China (30460125)
文摘Objective:To investigate the ectoparasite communities on Tupaia belangeri (T. belangeri) in the surrounding areas of Erhai Lake and their potential importance in medical and veterinary science. Methods: Different locations in surrounding areas of Erhai Lake in Yunnan were selected as the investigation spots. T. belangeri were randomly captured with baited mouse cages and then brought to the laboratory for identification according to their body shape, size and color, and measurements such as the lengths of their body, ear and hind feet. Ectoparasites on them were collected and identified to determine the species under microscope after specimens were mounted on slides, with the clearing and drying process completed. The constituent ratio (C), prevalence (P) and average ectoparasite abundance (A) were used to test the prevalence and density of ectoparasites; Non-parametric Mann-Whitney U-tests were used to test the differences in the abundance and richness of ectoparasites between female and male hosts; Spearman correlation analysis was used to analyze the relationship between ectoparasites and the body parameters of hosts. Results: There was very high species diversity of ectoparasites found on T. belangeri and some species of ectoparasites were reported associated with human diseases. A high proportion (88%) of 107 T. belangeri was found to be infested with ectoparasites. A total of 75 species of ectoparasites on them were collected, including 58 species of chigger mite, 12 species of gamasid mite, 4 species of flea and 1 species of sucking louse. Within this ectoparasite complex, 13 species were previously reported to be vectors of human disease agents. Non-parametric Mann-Whitney U-tests showed significant differences between male and female T. belangeri in terms of the abundance and richness of total ectoparasites. However, the species richness and abundance of chigger mites, sucking lice, fleas or gamasid mites presented no significant differences between male and female hosts. Spearman correlation analysis showed that none of correlations were observed between host body parameters with abundance and richness of total ectoparasites, chigger mites, suckinglice, fleas or gamatid mites. Conclusion: The results suggest that there is very high species diversity of ectoparasites on T. belangeri, consisting mainly of communities of sucking lice, fleas, chiggers and gamasid mites.
基金financially supported by the National Natural Scientific Foundation of China(Grant No.32160254)Natural Scientific Foundation of China(Grant No.31760118)+1 种基金Yunnan Ten Thousand Talents Plan Young&Elite Talents Project(YNWR-QNRC-2019-047)Yunnan Provincial Middle-Young Academic and Technical Leader candidate(2019HB013).
文摘Physiological adaptation of tree shrews(Tupaia belangeri)to changing environmental temperature has been re-ported in detail.However,the T.belangeri origin(mainland or island),population history,and adaptation to histor-ical climate change remain largely unknown or controversial.Here,for thefirst time,we sequenced the simplified genome of 134 T.belangeri individuals from 12 populations in China and further resequenced one individual from each population.Using population genomic approaches,wefirst observed considerable genetic variation in T.be-langeri.Moreover,T.belangeri populations formed obvious genetic structure and reflected different demographic histories;they generally exhibited high genetic diversity,although the isolated populations had relatively low ge-netic diversity.The results presented in this study indicate that T.b.modesta and T.b.tonquinia were separated recently and with a similar population dynamics.Second,physical barriers rather than distance were the driving factors of divergence,and environmental heterogeneity may play an important role in genetic differentiation in T.belangeri.Moreover,our analyses highlight the role of historical global climates in the T.belangeri population dynamics and indicate that the decrease of the T.belangeri population size may be due to the low temperature.Finally,we identified the olfaction-associated adaptive genes between different altitude populations and found that olfactory-related genes of high-altitude populations were selectively eliminated.Our study provides demographic history knowledge of T.belangeri;their adaption history offers new insights into their evolution and adaptation,and provides valuable baseline information for conservation measures.
文摘目的对丙型病毒性肝炎（hepatitis C virus，HCV）小动物模型树嗣的干扰素刺激基因15（interferon stimulated gene15，ISG15）分子的全长eDNA序列进行克隆及分子生物学功能分析，为树晌模型在HCV感染天然免疫中的研究提供分子生物学基础。方法根据Genbank中灵长类及其他哺乳类动物ISG15分子序列保守区设计引物，使用SmarterRace方法扩增树购ISG15全长序列。在序列两端设计特异性引物，引入酶切位点，进行全长片段扩增。全长PCR产物纯化回收后连接至pMD18-T载体，构建重组质粒pMD18-T-tbISG15。对重组质粒进行酶切鉴定、测序。对序列进行同源性及种系进化，同时使用SWISSMODEL同源建模方法进行蛋白二级、三级结构预测及功能分析。结果获得树购ISG15全长序列共687bp，编码157个氨基酸。树晌ISG15与其他哺乳动物ISG15高度同源，与人的核苷酸及氨基酸序列同源性分别高达72．99％及71．34％，核苷酸及氨基酸序列进化树分析均显示树嗣种系最为接近灵长类动物。结构域分析提示：树嗣ISG15主要由两个类泛素样结构域构成。软件预测所得树购ISG15三维结构与人ISG15三维结构高度相似，且具有类泛素样三维结构，动力学检测提示本试验预测的树嗣ISG15三维结构稳定，可信度高。结论对树胸ISG15的克隆进一步完善了对树购模型的认识。为进一步在体内研究HCV感染的天然免疫机制奠定了分子生物学基础。
基金This work was supported by the National Science and Technology Major Projects for Major New Drugs Innovation and Development(No.2018ZX09711003-005-003)the National Science and Technology Major Project of Infectious Diseases(No.2017ZX10304402)+2 种基金the National Natural Science Foundation of China(No.81871316,31730029)President Fund of Xiamen University(No.20720190124)The funders had no role in the study design,data collection and analysis,decision to publish,or preparation of the manuscript.
文摘The human liver chimeric mouse is a milestone animal model for hepatitis B virus(HBV)infection.Such mice with primary human hepatocyte(PHH)transplantation are adequate to support chronic HBV infection for several weeks and to evaluate antiviral drugs.However,the drawbacks of PHHs include lack of available donors,poor expansion in vitro and ethical issues that limit the application of human liver chimeric mice,necessitating the search for alternatives.Here,we transplanted primary tupaia hepatocytes(PTHs)into the livers of immunodeficient mice and achieved high liver chimerism within six weeks.These tupaia liver chimeric mice are adequate to support chronic infection of the four common HBV genotypes A,B,C and D for 36 weeks,as well as evaluate of antiviral drugs,including hepatitis B immune globulin(HBIG),monoclonal antibody and nucleoside analogues(NAs),for preventative therapy and treatment post infection.In conclusion,the tupaia liver chimeric mouse model provides a convenient,efficient and stable animal model for chronic HBV infection and long-term drug evaluation.
