Evidence for a porcine respiratory coronavirus, antigenically similar to transmissible gastroenteritis virus, in the United States

A respiratory variant of transmissible gastroenteritis virus (TGEV), designated PRCV-Ind/89, was isolated from a swine breeding stock herd in Indiana. The virus was readily isolated from nasal swabs of pigs of different ages and induced cytopathology on primary porcine kidney cells and and on a swine testicular (ST) cell line. An 8-week-old pig infected oral/nasally with the respiratory variant and a contact pig showed no signs of respiratory or enteric disease. These pigs did not shed virus in feces but did shed the agent from the upper respiratory tract for approximately 2 weeks. Baby pigs from 2 separate litters (2 and 3 days old) also showed no clinical signs following oral/nasal inoculation with PRCV-Ind/89. In a third litter, 5 of 7 piglets (5 days old) infected either oral/nasally or by stomach tube developed a transient mild diarrhea with villous atrophy. However, virus was not isolated from rectal swabs or ileal homogenates of these piglets, and viral antigen was not detected in the ileum by fluorescent antibody staining even though the virus was easily recovered from nasal swabs and lung tissue homogenates. Swine antisera produced against PRCV-Ind/89 or enteric TGEV cross-neutralized either virus. In addition, an anti-peplomer monoclonal antibody, 4F6, that neutralizes TGEV also neutralized the PRCV-Ind/89 isolate. Radioimmunoassays with a panel of monoclonal antibodies indicated that the Indiana respiratory variant and the European PRCV are antigenically similar.     

The Prevalence and Prevention of PEDV,TGEV and PoRV in Porcine Viral Diarrhea

PEDV,TGEV and PoRV are the main pathogenies of porcine viral diarrhea that endanger the healthy development of swine industry,which are characterized by vomiting,severe diarrhea and dehydration, the morbidity and mortality of suckling piglets is higher,and the incidence of diarrhea is lower in replacement gilts,sows or fattening pigs. Especially since 2010, the prevalence of PEDV mutant strains has had a serious impact on the global swine industry,but the control measures can not been able to cope with the new epidemic situation. In recent years, many new techniques or methods have been applied to the control of swine viral diarrhea caused by PEDV, TGEV and PoRV. Based on the analysis of genetic variation and epidemic status of three kinds of viruses,this paper reviewed the prevention and control measures from the aspects of feeding management,immunization prevention, traditional Chinese medicine therapy,interference therapy,specific therapy and return therapy.     

猪病毒性腹泻分子流行病学调查

为了解近年来中国猪病毒性腹泻的发生现状,于2011年4月至2012年4月利用多重RT-PCR方法对采集于13个省市的猪腹泻样品和临床健康的样品进行了检测,结果显示,腹泻猪群样品中TGEV阳性率为2.65%,PEDV阳性率为24.49%,ARV阳性率为3.20%;在肠道组织样品中,TGEV的阳性率为3.11%,PEDV为14.83%,ARV为1.67%;粪便样品中,TGEV的阳性率为2.80%,PEDV为28.42%,ARV为4.86%;母猪(所产仔猪腹泻)乳汁中,TGEV阳性率为1.08%,PEDV为31.89%,ARV为0.54%。健康猪群样品中,保育与育肥阶段猪中PEDV阳性率为2.13%,ARV阳性率为1.42%;哺乳仔猪中ARV阳性率为12.86%。由此可知,目前中国猪病毒性腹泻以PED为主要病因,PEDV和TGEV在幼龄猪群中存在隐性感染现象,且3种病毒性腹泻均可通过母乳传播病毒。     

2016－2019年广西部分猪场仔猪病毒性腹泻的流行病学调查

本研究旨在了解引起仔猪腹泻的主要病毒性病原感染情况及流行特点,为有效防控广西仔猪腹泻提供科学依据。试验采用RT-PCR/PCR检测方法对2016年3月至2019年2月广西14个地级市366个规模猪场送检的914份仔猪腹泻病料样品进行猪流行性腹泻病毒（PEDV）、猪轮状病毒（PoRV）、猪传染性胃肠炎病毒（TGEV）、猪Delta冠状病毒（PDCoV）、猪瘟病毒（CSFV）、猪繁殖与呼吸综合征病毒（PRRSV）、猪圆环病毒2型（PCV2）和猪伪狂犬病病毒（PRV）检测,并分析其阳性率在不同年份、季节、地区之间的差异及病原混合感染情况。调查结果显示,PEDV、PDCoV、PRRSV、PoRV、PCV2、CSFV、PRV均存在不同程度的感染,其样品平均阳性率分别为59.74%、8.32%、7.77%、4.92%、3.72%、3.28%和2.08%,未检测出TGEV;PEDV已无明显季节性,一年四季均高发,即使在炎热的夏季,在感染猪群中也持续存在,PDCoV有明显的季节性,多发生在冬春寒冷季节;广西仔猪腹泻PEDV阳性率高,单一感染高达50.55%,仔猪腹泻混合感染情况也较多,其中以二重感染情况较为常见,同时也存在四重感染现象。结果表明,广西腹泻仔猪存在7种病毒性病原不同程度感染情况,其中以PEDV导致的仔猪病毒性腹泻尤为严重,新发PDCoV阳性率仅次于PEDV,需重视并加强对新发PDCoV的防控。     

猪流行性腹泻病毒细胞受体研究进展

猪流行性腹泻病毒主要引起仔猪腹泻,发病率及病死率极高,严重影响养猪业的发展。而猪流行性腹泻病毒要感染仔猪,必须与宿主细胞表面的受体结合。目前已有研究证实,猪流行性腹泻病毒的细胞表面受体为猪氨基肽酶N(pAPN)。为了能更好的揭示pAPN在病毒感染过程中的机制,学者针对pAPN的结构、功能以及与病毒的感染结合区域进行了系统的研究,并开展了筛选pAPN结合短肽的工作,以此来探索病毒受体的阻断剂。为进一步研究猪流行性腹泻病毒感染机制及其细胞受体的作用提供参考,论文就目前pAPN最新研究进展进行了综述。     

Monoclonal antibody-based immunohistochemical detection of porcine epidemic diarrhea virus antigen in formalin-fixed, paraffin-embedded intestinal tissues.

An immunohistochemistry technique was developed for the diagnosis of porcine epidemic diarrhea virus (PEDV). The technique was tested on formalin-fixed, paraffin-embedded intestinal tissues from piglets naturally infected with PEDV. Five different monoclonal antibodies (MAbs) were tested in this study. PEDV antigen was consistently detected in the PLP (4% paraformaldehyde, 100 mM L-lysine dihydrochloride, 10 mM sodium m-periodate in phosphate-buffered saline)-fixed PEDV-infected Vero cells or formalin-fixed, paraffin-embedded intestinal tissues from piglets naturally infected with PEDV. The C9-2-2 MAb gave the strongest reactivity and least background staining, detecting 10 of 10 infected pigs. The positive reaction was cytoplasmic. Positive enterocytes were distributed over the tip and along the sides of atrophied or fused villi in the jejunum and ileum. Positive-staining cells were not detected in the crypts. No staining was observed in cecum and colon. No positive cells were observed when the C9-2-2 MAb was reacted with the tissue sections from noninfected piglets or from transmissible gastroenteritus virus (TGEV)- and rotavirus-infected piglets. The selected anti-PEDV MAbs tested on formalin-fixed, paraffin-embedded tissue sections are useful for diagnosis when virus isolation is not available. This method would be of particular value in countries where both PEDV and TGEV are epizootic and would aid in differentiating between PEDV and TGEV infection.     

猪传染性胃肠炎与流行性腹泻病毒二重RT-PCR 检测方法的建立及应用

为建立猪传染性胃肠炎病毒(TGEV)与流行性腹泻病毒(PEDV)的快速鉴别诊断方法,本研究根据GenBank已登录的TGEV核蛋白(N)基因和PEDV膜蛋白(M)基因保守区域序列分别设计了1对特异性引物,以TGEV和PEDV混合总RNA为反转录模板,建立了TGEV和PEDV的二重RT-PCR检测方法,并进行了特异性、敏感性和重复性试验;利用所建立的检测方法对临床疑似样品进行了应用检测,并对检测到的阳性样品进行克隆测序。结果表明,成功建立了TGEV和PEDV二重RT-PCR检测方法,该方法的检测灵敏度最低极限为10 TCID₅₀/mL病毒含量,重复性好,特异性强,可特异性地扩增TGEV和PEDV细胞培养物,但对ST细胞和其他7种病原对照扩增不出任何条带;对22份临床疑似TGEV和PEDV感染样品检测结果与测序结果完全一致。本研究成功建立了TGEV与PEDV二重RT-PCR检测方法,可适用于猪传染性胃肠炎和流行性腹泻病的快速鉴别诊断。     

Bovine viral diarrhea virus isolated from fetal calf serum enhances pathogenicity of attenuated transmissible gastroenteritis virus in neonatal pigs.

A bovine viral diarrhea virus (BVDV-C) was isolated from swine tissue culture cells used to attenuate the transmissible gastroenteritis virus (TGEV) after 68 passes. Piglets given a pure culture of BVDV-C developed clinical signs similar to those of a mild TGEV infection and recovered by 10 days postexposure. Villous blunting and fusion was observed in the small intestine, and a lymphocyte depletion was observed in Peyer's patches in the ileum. Piglets given a combination of BVDV-C and attenuated TGEV developed clinical signs similar to those of a virulent TGEV infection and were euthanized. The combined infection induced a generalized lymphocyte depletion throughout the lymphatic system and villous atrophy in the intestinal tract. Piglets exposed to a another type I strain of BVDV (NY-1) either alone or in combination with the attenuated TGEV had mild clinical signs similar to those of a TGEV infection. Moderate villous atrophy in the ileum and a lymphocyte depletion in the mesenteric lymph node were observed in these piglets postmortem. The data indicate a potential problem for diagnostic laboratories in relation to a diagnosis of virulent TGEV infections and in the field for young piglets exposed to a BVDV-contaminated TGEV vaccine.     

猪流行性腹泻病毒、传染性胃肠炎病毒、A群轮状病毒和嵴病毒多重RTPCR检测方法的建立及临床应用

本研究建立了可同时检测猪流行性腹泻病毒（Porcineepidemicdiarrheavirus,PEDV）、传染性胃肠炎病毒（TransmissiblegastroenteritisVirus,TGEV）、A群轮状病毒（GroupArotavirus,GARV）和猪嵴病毒（Porcinekobu—virus）的多重RT—PCR方法。检测中,建立的多重RT—PCR方法能够检测到500Pg的TGEV、PEDV、GARV和猪嵴病毒等量混合RNA模板,与常规的单一RT—PCR检测结果基本相同（检测TGEV、PEDV、GARV和猪嵴病毒的灵敏性分别为1000A、1000／6、93．33％和96．67％,特异性均为i00％）。结果表明,建立的多重RTPCR方法敏感性和特异性良好,可作为临床上猪病毒性腹泻病因快速、高效的诊断工具。应用该方法对2010—2012年华中地区190份腹泻仔猪样本进行检测,PEDV、TGEV、GARV和猪嵴病毒的阳性率分别为62．11％、0．53％、7．37％和82．11％。混合感染方面,PEDV和猪嵴病毒混合感染率为47．89％,PEDV和GARV混合感染率为4．74％,GARV和猪嵴病毒混合感染率为7．37％,PEDV、GARV和猪嵴病毒混合感染率为4．74％,未发现TGEV与其它3种病毒的混合感染情况。另外,有27份样本中仅检出PEDV（14．21％）,57份样本只检出猪嵴病毒（30％）。分析表明,我国自2010年底大面积暴发的病毒性腹泻是多病原混合感染造成的,主要病原为PEDV,猪嵴病毒在其中所起作用尚待进一步验证和研究。     

一例高致病性猪蓝耳病、猪流行性腹泻混合感染的诊断

2018年11月,湖南郴州某规模化猪场发生2~3日龄仔猪呕吐、腹泻为主的疫情,发病率70%,死亡率80%,为确定引起仔猪腹泻的原因,从发病猪群中采集2头病死猪的小肠、肺脏、淋巴结等组织进行PCR检测及基因测序分析。检测结果显示猪流行性腹泻和猪蓝耳病均为阳性,猪传染性胃肠炎、猪轮状病毒均为阴性。测序分析显示所检测到的猪蓝耳病病毒与JXA1毒株高度同源,为高致病性毒株,检测到的猪流行性腹泻病毒为变异毒株,隶属于GⅡ-b群。综合分析,引起此次新生仔猪大批死亡系变异猪流行性腹泻病毒混合感染高致病性猪蓝耳病所致。     

Several enteropathogens are circulating in suckling and newly weaned piglets suffering from diarrhea in the province of Villa Clara, Cuba

Intestinal contents of suckling (n?=?45) and newly weaned (n?=?45) piglets, suffering from diarrhea in the province of Villa Clara in Cuba, were tested for viral, bacterial, and parasitic enteropathogens from May to June 2008. At least one enteropathogen was identified in 53.3 % of piglets and enterotoxigenic Escherichia coli (ETEC; 25.6 %) was the major pathogen; mostly STa⁺/STb⁺ or F4⁺/STa⁺/STb⁺ ETEC were isolated. The overall occurrence of the rest of pathogens was 10 % for transmissible gastroenteritis virus (TGEV) and Cryptosporidium parvum, 6.7 % for rotavirus A and Isospora suis, 5.6 % for α-toxigenic Clostridium perfringens, 3.3 % for verotoxigenic E. coli (VTEC), and 2.2 % for Salmonella enterica subspecies enterica serovar Newport. TGEV and α-toxigenic C. perfringens were only identified in suckling piglets, while Salmonella Newport and VTEC were only detected in weaned pigs. Porcine epidemic diarrhea virus (PEDV), β-toxigenic C. perfringens, Eimeria spp., and helminths were not identified. Eight kinds of mixed infections were detected in 25 % of enteropathogen positive piglets. ETEC was present in 10 of 12 mixed infections, and TGEV infections were never combined. This survey demonstrates that several enteropathogens are circulating in piggeries located in the province of Villa Clara in Cuba, and that is necessary to improve surveillance, prevention, and control of enteric infections in order to increase production efficiency.     

利用CRISPR/Cas9编辑系统构建pAPN基因敲除的IPI-2I细胞系

试验旨在采用CRISPR/Cas9技术获得猪氨基肽酶N（porcine aminopeptidase N,pAPN）基因敲除的猪回肠上皮（immortal pig intestinal-2I,IPI-2I）细胞系,进而在细胞水平上研究pAPN在冠状病毒入侵过程中的作用及相互作用机制。本研究将已构建好的靶向pAPN基因第2外显子上的2个sgRNA载体（pX330-GFP-g3和pX330-RFP-g5）共转染IPI-2I细胞。转染48 h后,荧光显微镜下观察IPI-2I细胞绿色荧光蛋白（green fluorescent protein,GFP）和红色荧光蛋白（red fluorescent protein,RFP）的发光情况,并用流式细胞仪收集带有GFP和RFP荧光标记的细胞,用来筛选单克隆细胞。然后取少量单克隆细胞提取DNA,用PCR扩增打靶区域附近的序列,测序鉴定其基因型,以获得pAPN基因敲除的IPI-2I细胞株。选择野生型和pAPN基因纯合片段敲除的IPI-2I细胞,提取细胞总蛋白,通过Western blotting检测pAPN基因敲除前后IPI-2I细胞中pAPN蛋白的表达。结果表明,转染48 h后,通过荧光显微镜可以观察到IPI-2I细胞中大部分细胞能够同时表达GFP和RFP,说明pX330-GFP-g3和pX330-RFP-g5质粒载体已经转入IPI-2I细胞。PCR和测序结果表明,共获得48株片段敲除单克隆细胞（片段敲除效率为15.5%）,其中16株为单等位基因敲除,32株为双等位基因敲除;在32株双等位基因敲除细胞中,有23株细胞为纯合片段敲除。Western blotting结果表明,pAPN基因纯合片段敲除的细胞中检测不到pAPN蛋白的表达。综上,本研究利用CRISPR/Cas9编辑系统成功构建了pAPN基因纯合敲除的IPI-2I细胞系,为阐明pAPN在冠状病毒入侵过程中的作用机制以及制备猪抗病新品种奠定了基础。     

血清复合制剂对仔猪腹泻综合征的疗效比较

为了获得有效治疗仔猪腹泻的新方法,本试验选择了几种引起仔猪腹泻的病原疫苗接种屠宰前60d左右的育肥猪,然后制备血清及血清制剂,按1∶1和1∶1.5将血清制剂与恩诺沙星配比应用于4个试验猪场共221头腹泻仔猪。结果显示:空白对照组、单一使用血清制剂组及单一使用恩诺沙星组的平均治愈率分别为55%、56%、73%;而复合制剂组1和复合制剂组2的治愈率分别为91%和100%。试验证明,将血清制剂与恩诺沙星按1∶1.5配比使用,其治愈率最高,可作为有效防控仔猪腹泻的首选方法。     

猪传染性胃肠炎病毒吉林株的分离鉴定及S基因的克隆与序列分析

利用PK-15细胞从临床上表现为腹泻症状的病死仔猪肠系膜淋巴结材料中分离获得1株病毒,对该病毒进行病毒形态学、PCR检测与动物回归试验等系统鉴定后,证实该分离株为猪传染性胃肠炎病毒(TGEV),命名为TGEVJL。利用PCR方法克隆出其S基因部分片段,并将该基因序列和推导的氨基酸序列与7个不同来源的TGEV毒株进行同源性和亲缘关系的比较分析,结果表明,各毒株间核苷酸和氨基酸的同源性分别为96.3%～99.3%和94.8%～98.8%;系统进化树结果表明,TGEVJL株与日本分离的TQ14毒株和西班牙分离的TOY56-165毒株亲缘较近,表明不同地区分离毒株的S基因差异不大。     

2017—2019年华东地区猪场主要病毒性腹泻病原调查

本研究旨在了解当前我国华东地区引起猪腹泻的主要病毒性腹泻病原的流行情况，为该地区更好地开展猪腹泻病毒的防控工作提供临床数据。分别采用RT-PCR检测方法对2017—2019年华东地区35个场别594份临床样品检测猪流行性腹泻病毒（porcine epidemic diarrhea virus，PEDV）、猪轮状病毒（porcine rotavirus，PoRV）、猪传染性胃肠炎病毒（transmissible gastroenteritis virus of swine，TGEV）、猪丁型冠状病毒（porcine deltacoronavirus，PDCoV）和猪萨佩罗病毒（porcine Sapelovirus，PSV）等5种猪的病毒性腹泻病原，并对其阳性率及混合感染情况进行统计分析。结果显示，2017—2019年的PEDV、PDCoV、PoRV、TGEV及PSV的平均阳性率为分别为62.6%（372/594）、27.9%（166/594）、16.8%（100/594）、13.3%（79/594）及3.87%（23/594），二重混合感染中以PEDV+PDCoV及PEDV+PoRV为主，平均混合感染率分别为22.4%（133/594）和13.3%（79/594）。三重感染中以PEDV+PoRV+PDCoV混合感染为主，感染率为7.58%（45/594），未见四重及五重病原混合感染。2017—2019年PEDV平均阳性检出率最高，且常与PoRV及PDCoV发生混合感染，是当前华东地区猪病毒性腹泻类疫病的防控重点。TGEV和PDCoV在健康育肥猪及母猪的检出率中占比较大，表明隐性感染变得普遍，值得养殖户关注。除此之外，PSV的检出率远低于其他4种腹泻病毒。总之，随着2018年后养殖场加强生物安全防控措施和猪群管理，各病原检出率和阳性场检出率呈现逐年下降趋势。     

Investigation on Pathogens of Major Viral Diarrhea in Pig Farms in East China from 2017 to 2019

To investigate the epidemic situation of viral diarrhea in East China in recent years, and provide epidemiological survey data for comprehensive prevention, 549 fecal samples were collected in 2017-2019 from diarrhea pigs in 5 cities of East China and RT-PCR were used to detect porcine epidemic diarrhea virus (PEDV), porcine rotavirus (PoRV), porcine transmissible gastroenteritis virus (TGEV), porcine deltacoronavirus (PDCoV) and porcine Sapelovirus (PSV), and statistical analysis of its positive rate and mixed infection was performed.The results showed that during 2017 and 2019, PEDV,PDCoV,PoRV,TGEV and PSV positive rate were 62.6% (372/594),27.9% (166/594),16.8% (100/594),13.3% (79/594), and 3.87% (23/594), respectively.In the double mixed infection, the average mixed infection rate of PEDV + PDCoV and PEDV + PoRV were the main ones, which were 22.4% (133/594) and 13.3% (79/594), respectively. Among the triple infections, the mixed infection rate of PEDV + PoRV + PDCoV was dominated by 7.58% (45/594), and there were no mixed infections of four and five pathogens. The highest positive detection rate of PEDV was during 2017 and 2019, and mixed infection with PoRV and PDCoV, which was currently the focus of prevention and control of viral diarrhea in East China. TGEV and PDCoV accounted for a large proportion of the detection rate of healthy fattening pigs and sows, indicating that recessive infections had become common and deserve attention from farmers. In addition, the detection rate of PSV was more lower than the other four diarrhea viruses. On the whole, since August 2018 that the farms strengthened biosecurity prevention and control measures, the detection rate of various pathogens and the detection rate of sites have shown a downward trend year by year.     

A survey of agents associated with neonatal diarrhea in Iowa swine including Clostridium difficile and porcine reproductive and respiratory syndrome virus.

This survey was undertaken to determine the relative frequency of agents that are currently associated with neonatal diarrhea in swine, including Clostridium difficile and porcine reproductive and respiratory syndrome virus (PRRSV). The subjects for this study were the first 100 live 1-7-day-old piglets submitted to the Iowa State University Veterinary Diagnostic Laboratory with a clinical signalment of diarrhea, beginning on January 1, 2000. The evaluation of each pig included bacterial culture of a section of ileum, 2 sections of jejunum, and a single section of colon; a fluorescent antibody test (FAT) or immunohistochemistry (IHC) for transmissible gastroenteritis virus (TGEV); ELISA's for rotavirus and C. difficile toxins; IHC for PRRSV; and microscopic examination of ileum, midjejunum, spiral colon, liver, spleen, and lung. Survey results demonstrate a decline in the relative number of diagnoses of TGEV, Escherichia coli, and Clostridium perfringens type C compared with retrospective data. The combined case frequency rate for these 3 pathogens dropped from 70% in 1988 to 21% in 2000. This survey also demonstrated the emergence of C. difficile as an important pathogen of neonatal swine. Clostridium difficle toxin was detected in the colon contents of 29% of the piglets, and at least 1 toxin-positive animal was identified in 55% of the cases. All 29 C. difficile toxin-positive piglets had mesocolonic edema, and colitis was observed in 21 of 29 toxin-positive animals. PRRSV-positive macrophages were detected in the lamina propria of intestinal villi by IHC in 10 piglets with diarrhea. In 6 of these cases, PRRSV was the only pathogen detected. Gross and microscopic lung lesions were not a reliable indicator of PRRSV infection in these neonatal pigs with diarrhea. The addition of tests for C. difficile and PRRSV to a routine neonatal diarrhea diagnostic protocol resulted in a significant increase in thediagnostic success rate on both individual animal and case bases.     

Study on Effect of Two Types of Compound Immunoglobulin Yolk Powder on Post-weaning Swine Diarrhea

For developing immunoglobulin yolk powder products to prevent and treat the diarrheal piglets, and appling the advanced technology of egg yolk antibody to the husbandry and aquaculture, two types of compound immunoglobulin yolk powder were used to prevent and treat the diarrhea piglets which were challenged with ETEC, PEDV and TGEV.The creep feed added with 0.4% typeⅠimmunoglobulin yolk powder was provided to experimental piglets.There was no death after challenging the piglets with ETEC or virus, only 5 minor diarrhea in piglets in the early infection.As feeding continuing, the 5 piglets diarrhea quickly brought under control, conversely, the piglets in negative control group were totally dead.The immunoglobulin yolk powder type Ⅱ mixed with GNS at the rate of 1∶3 was used to cure the diarrhea piglets, 20 mL per piglet and 2 times a day.After curing for 3 days, the diarrhea was greatly improved, for 5 days the piglets diarrhea was basic recovery, and the survival rate could reach 84% to 88%.Dectected the infected of surviving piglets, compared to the drug treatment control group, prevention group had the lowest pathogen amounts, and the drug treatment control group had the most serious infected.     

生物信息学软件预测猪氨基肽酶N功能与结构

[目的]研究猪δ冠状病毒(PDCoV)入侵宿主细胞的感染机制,对其特异性受体猪氨基肽酶N基因编码蛋白功能与结构进行分析.[方法]通过多种生物信息学软件对pAPN蛋白的理化特性、亲疏水性、信号肽、跨膜区、糖基化位点、抗原表位、功能结构域及结构特征等进行预测分析.[结果]pAPN基因总长为2892 bp,编码963个氨基酸...