WO2021098684A1 - 一种表达E2-crimson的重组伪狂犬病毒及其制备方法和应用 - Google Patents

一种表达E2-crimson的重组伪狂犬病毒及其制备方法和应用 Download PDF

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WO2021098684A1
WO2021098684A1 PCT/CN2020/129494 CN2020129494W WO2021098684A1 WO 2021098684 A1 WO2021098684 A1 WO 2021098684A1 CN 2020129494 W CN2020129494 W CN 2020129494W WO 2021098684 A1 WO2021098684 A1 WO 2021098684A1
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pseudorabies virus
crimson
recombinant pseudorabies
vector
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姜璐
路中华
刘太安
陈晔菲
纪文静
柏艳阳
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Shenzhen Institute of Advanced Technology of CAS
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  • the invention belongs to the technical field of neural engineering, and relates to a recombinant pseudorabies virus expressing E2-crimson, and a preparation method and application thereof.
  • the nervous system is a complex neural network composed of a large number of neurons connected to each other. Revealing the connection between various functional neurons is a basic problem in the field of neuroscience research, which is of great significance for understanding the normal function of the brain and the pathological mechanism of neurological diseases.
  • Neural circuit tracing technology for the purpose of depicting the connection of neurons is the key to current brain science research. It ranks second among the nine priority funding projects of the American Brain Project, and is also the basis for the implementation of the European Brain Project and the Korean Brain Project. core.
  • Existing neural circuit tracers include fluorescent dyes, biotin, and neurotropic viruses. Among them, the diffusion of traditional tracers represented by fluorescent dyes and biotin in nerve tissues does not have strict synaptic specificity, and its application in tracing nerve synaptic connections has great limitations.
  • Neurotropic viruses are widely used in neural circuit tracing because they can infect neurons and transmit along nerve synapses. Compared with traditional tracers, neurotropic viruses have the characteristics of carrying foreign genes, self-replicating, signal expansion, and trans-synaptic transmission. It is currently the most commonly used neural circuit tracer.
  • the commonly used neural network tracer viruses include herpes simplex virus, rabies virus and vesicular stomatitis virus.
  • the pseudorabies virus PRV belonging to the herpes simplex virus family is one of the most promising neural tracing tool viruses due to its reverse trans-synaptic transmission, the ability to carry large fragments of foreign genes, and high biological safety. .
  • the wild-type PRV-Becker strain can be traced bidirectionally along the synapse.
  • the modified attenuated strain PRV-Bartha can only transmit across the synapse in the reverse direction.
  • Most of the viruses used in neural circuit tracing are in the attenuated strain PRV- Recombinant PRV modified on the basis of Bartha, so it can only be used for reverse transfer marking.
  • PRV viruses expressing green fluorescent protein or red fluorescent protein, such as PRV152 expressing green fluorescent protein EGFP, PRV600 and PRV613 expressing red fluorescent protein DsRed, and PRV614 expressing red fluorescent protein mRFP.
  • DsRed is the first red fluorescent protein to be discovered, cloned from Discosoma coral, and a series of monomeric red fluorescent protein mRFPs were subsequently developed.
  • PRV carrying the red fluorescent protein gene is PRV614, which is usually injected into specific brain regions of mice alone or together with PRV of other colors to study related neural circuits.
  • the existing recombinant PRV viruses expressing red fluorescent protein generally have problems such as weak fluorescence intensity, long maturation time, and poor transmission ability, which are not conducive to neural network tracing research. Therefore, it is necessary to develop a new recombinant PRV carrying red fluorescent protein to make the expressed red fluorescent protein brighter and faster to mature, and to be better used for neural circuit tracing.
  • the present invention provides a recombinant pseudorabies virus expressing E2-crimson and a preparation method and application thereof.
  • the red fluorescent protein E2-crimson used in the recombinant pseudorabies virus has high fluorescence intensity. , Fast maturation time, strong transmission ability, has a wide range of application prospects in the field of neural circuit tracing.
  • the present invention adopts the following technical solutions:
  • the present invention provides a recombinant pseudorabies virus vector, the recombinant pseudorabies virus vector including E2-crimson gene;
  • the nucleic acid sequence of the E2-crimson gene is shown in SEQ ID NO:1;
  • the nucleic acid sequence shown in SEQ ID NO:1 is:
  • E2-Crimson is the first fluorescent protein to be effectively excited by a standard far-red laser.
  • E2-crimson is used as the red fluorescent protein.
  • the extinction coefficient is as high as 126000M -1 cm -1 , the medium quantum yield is 0.23, and the brightness is Strong, the fluorescence intensity is 2.3 times that of mRFP, and the maturation time is three times faster than mRFP, which shortens the experiment time.
  • E2-crimson has the maximum absorption at 611nm and the maximum emission at 646nm. Compared with mRFP, it has a significant red shift and transmission. It has strong ability and has significant application potential in the field of neural circuit tracing.
  • the recombinant pseudorabies virus vector uses the pBB5 vector as an empty vector, and the E2-crimson gene is ligated into the pBB5 vector to obtain the recombinant pseudorabies virus vector pBB5-E2-crimson.
  • the pBB5 vector containing the homologous recombination arm of the PRV152 genome is selected to connect the E2-crimson gene.
  • the present invention provides a method for preparing the recombinant pseudorabies virus vector as described in the first aspect, and the preparation method includes the following steps:
  • restriction enzyme NsiI-HF to treat pCMVGF-E2-crimson vector, while using restriction enzyme pstI-HF to treat pBB5 vector;
  • the two digested products are recovered and connected to obtain the recombinant pseudorabies virus vector pBB5-E2-crimson.
  • the restriction endonucleases pstI-HF and NsiI-HF are homologous to each other, that is, the sticky ends produced by the two kinds of endonuclease digestion can be complemented and paired, and the restriction enzymes pstI-HF and NsiI-HF are treated with
  • the vectors can be joined by ligase to form a new recombination vector, and the E2-crimson gene is connected between the two homologous recombination arms.
  • the present invention provides a recombinant pseudorabies virus, which includes the recombinant pseudorabies virus vector as described in the first aspect.
  • the present invention provides a method for preparing the recombinant pseudorabies virus as described in the third aspect, the preparation method comprising the following steps:
  • step (1) linearize the recombinant vector pBB5-E2-crimson with restriction enzyme SpeI.
  • SpeI is next to the 5'homologous recombination arm.
  • 5' The 3'homologous recombination arm and the E2-crimson gene in the middle are still connected together without being separated by restriction enzymes, which ensures the smooth progress of the homologous recombination with the PRV152 genome in the next step.
  • the PRV152 virus is used as the parent, because PRV152 expresses green fluorescent protein EGFP, while PRV-E2-crimson has red fluorescence. The difference in fluorescence color is used to distinguish the parental PRV152 from recombinant PRV-E2-crimson under a fluorescence microscope. .
  • the method for extracting the PRV152 viral genome in step (2) includes:
  • the PRV152 virus is used to infect the host cell, so that the PRV152 virus is amplified in the host cell; the host cells are collected and subjected to cell lysis; DNA extraction is used to obtain the PRV152 virus genome.
  • the host cell in step (3) includes any one or a combination of at least two of 293 cells, 293T cells or 293F cells.
  • step (3) the step of screening and purifying the recombinant pseudorabies virus is further included.
  • the E2-crimson gene is connected to the PRV152 genome by the method of homologous recombination to construct a structure capable of stably expressing the red fluorescent protein E2-crimson, self-replicating, and reverse display.
  • the main steps of tracking the PRV-E2-crimson virus are as follows:
  • the present invention provides a neural network tracer, which comprises the recombinant pseudorabies virus vector as described in the first aspect and/or the recombinant pseudorabies virus as described in the third aspect .
  • this aspect provides a kit comprising the recombinant pseudorabies virus vector as described in the first aspect, the recombinant pseudorabies virus as described in the third aspect, or the recombinant pseudorabies virus as described in the fifth aspect Neural network tracer.
  • the present invention provides a recombinant pseudorabies virus vector as described in the first aspect, a recombinant pseudorabies virus as described in the third aspect, a neural network tracer as described in the fifth aspect, or a recombinant pseudorabies virus as described in the fifth aspect.
  • the present invention has the following beneficial effects:
  • the recombinant PRV-E2-crimson of the present invention uses E2-crimson as the red fluorescent protein, the fluorescence intensity is 2.3 times that of mRFP, the maturation time is three times faster than mRFP, and the experiment time is shortened.
  • E2-crimson has a maximum at 611nm. Absorption, maximum emission at 646nm, obvious red shift relative to mRFP, strong transmission ability;
  • the recombinant PRV-E2-crimson of the present invention can efficiently reversely label neural circuits in the mouse striatum, with high expression efficiency and strong infectivity;
  • the recombinant PRV-E2-crimson of the present invention has broad application prospects in the field of neural circuit tracing.
  • Figure 1 is a map and preparation flow chart of the recombinant vector pBB5-E2-crimson
  • Figure 2(A) shows the results of the first round of screening by PRV-E2-crimson
  • Figure 2(B) shows the results of the second round of screening by PRV-E2-crimson
  • Figure 2(C) shows the results of PRV-E2-crimson.
  • Figure 3 shows the expression of PRV-E2-crimson in the mouse striatum and its upstream loop.
  • the PRV152 genome is used as the recombination parent. Therefore, when constructing the recombinant vector, the pBB5 vector containing the homologous recombination arm of the PRV152 genome is used to connect the E2-crimson gene. The steps are as follows:
  • the pBB5 vector was treated with restriction endonuclease pstI-HF (NEB), while the pCMVGF-E2-crimson vector was treated with restriction endonuclease NsiI-HF (NEB), and digested at 37°C for 3h,
  • the digestion system is shown in Table 1-1 and Table 1-2.
  • the ligation premix (2 ⁇ ligation premix, TAKARA) is used for ligation at 16°C overnight.
  • the system is shown in Table 1-3.
  • the successfully connected pBB5-E2-crimson vector is obtained.
  • recombinant vector pBB5-E2-crimson 5 ⁇ g of recombinant vector pBB5-E2-crimson was linearized with restriction endonuclease SpeI at 37°C for 2h. The system was shown in Table 2. After restriction enzyme digestion, DNA was extracted and purified with phenol and chloroform to obtain linearized recombinant vector.
  • PRV152 virus As the parent, because PRV152 expresses green fluorescent protein EGFP, and PRV-E2-crimson has red fluorescence. The difference in fluorescence color is used to distinguish between parental PRV152 and recombinant PRV-E2-crimson under a fluorescence microscope.
  • the extraction steps of PRV152 genome are as follows:
  • the PRV152 virus is used to infect the host cell pig kidney cell PK15, so that the PRV152 virus is amplified in the host cell;
  • the calcium transfection method is used to introduce the linearized pBB5-E2-crimson and PRV152 genomes into 293T cells. Under the action of recombinase, the two complete the homologous recombination process with the aid of the homologous recombination arm to obtain a recombinant PRV. -E2-crimson.
  • transfection reagents include A (12.5 ⁇ L 2M CaCl 2 , 1 ⁇ g PRV152 genomic DNA, 1 ⁇ g linearized pBB5-E2-crimson DNA, 2 ⁇ g calf thymus DNA, filled with ddH 2 O to 100 ⁇ L) and B (100 ⁇ L 2 ⁇ HBS);
  • PRV152 genomic DNA and linearized pBB5-E2-crimson have long homology arms and short homology arms.
  • 293T cells contain recombinase. Under the action of recombinase, the two exchange components in the homology arms to complete homologous recombination. .
  • the formed virus products include both the successfully recombined red fluorescent PRV-E2-crimson and the green fluorescent parent PRV152.
  • the successfully recombined PRV-E2-crimson was obtained through screening, specifically:
  • mice Three 8-week-old C57 mice were selected and injected 2 ⁇ L of PRV-E2-crimson with a titer of 2 ⁇ 10 9 pfu/mL into the striatum of the mice. Three days later, the mice showed obvious signs of PRV infection, and they moved slowly. Loss of appetite and weight loss, the mice were perfused to take the brains, sliced and stained using immunohistochemical methods, and observed the results of the brain slices.
  • STR striatum
  • Amy amygdala
  • STN subthalamic nucleus
  • VTA ventral tegmental area
  • the recombinant PRV-E2-crimson of the present invention uses E2-crimson as the red fluorescent protein, the fluorescence intensity is 2.3 times that of mRFP, and the maturation time is three times faster than mRFP, which shortens the experimental time.
  • E2-crimson has the maximum absorption at 611nm and the maximum emission at 646nm. Compared with mRFP, it has a significant red shift and strong transmission ability; the recombinant PRV-E2-crimson can be found in the mouse striatum. Efficiently reversely label neural circuits, with high expression efficiency and strong infection ability, and has a wide range of application prospects in the field of neural circuit tracing.

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Abstract

一种表达E2-crimson的重组伪狂犬病毒及其制备方法和应用，所述表达E2-crimson的重组伪狂犬病毒的重组载体为包括E2-crimson基因的pBB5-E2-crimson载体。重组伪狂犬病毒中采用的红色荧光蛋白E2-crimson荧光强度高、成熟时间快、透射能力强，对细菌和哺乳动物细胞无细胞毒性，制备的重组伪狂犬病毒在小鼠纹状体中可以高效地逆向标记神经环路，表达效率高，侵染能力强，在神经环路示踪领域具有广泛的应用前景。

Description

一种表达E2-crimson的重组伪狂犬病毒及其制备方法和应用 技术领域
本发明属于神经工程技术领域，涉及一种表达E2-crimson的重组伪狂犬病毒及其制备方法和应用。
背景技术
神经系统是由数目众多的神经元相互连接而构成的复杂神经网络。揭示各个功能神经元之间的连接方式是神经科学研究领域的基本问题，对于理解大脑正常功能和神经系统疾病的病理机制具有十分重要的意义。以描绘神经元连接方式为目的的神经环路示踪技术是目前脑科学研究的关键，位列美国脑计划九个优先资助项目的第二名，也是欧洲脑计划实施的基础和韩国脑计划的核心。
现有的神经环路示踪剂包括荧光染料、生物素和嗜神经病毒等。其中，以荧光染料和生物素为代表的传统示踪剂在神经组织中的扩散没有严格的突触特异性，应用于示踪神经突触连接具有很大的局限性。
嗜神经病毒由于其能够感染神经元并沿神经突触传递，在神经环路示踪中得到了广泛的应用。与传统的示踪剂相比，嗜神经病毒具有可携带外源基因、能够自我复制进行信号扩大、跨突触传播等特性，是目前最常用的神经环路示踪剂。现常用的神经网络示踪病毒包括单纯疱疹病毒、狂犬病毒和水泡口炎病毒等。其中，隶属于单纯疱疹病毒家族的伪狂犬病毒PRV由于其逆向跨突触传播、可携载大片段外源基因和生物安全性高等特点，是当前最具开发潜力的神经示踪工具病毒之一。野生型PRV-Becker株可以沿突触双向示踪，改造后的减毒株PRV-Bartha只能逆向跨突触传递，应用于神经环路示踪中的大多数病毒是在减毒株PRV-Bartha基础上改造的重组PRV，因此只能进行逆向传递标记。
目前，已有多种表达绿色荧光蛋白或红色荧光蛋白的重组PRV病毒，例如表达绿色荧光蛋白EGFP的PRV152，表达红色荧光蛋白DsRed的PRV600和PRV613、以及表达红色荧光蛋白mRFP的PRV614。DsRed是第一个被发现的红色荧光蛋白，克隆自Discosoma珊瑚，后续发展了一系列单体红色荧光蛋白mRFP。目前常用的携带红色荧光蛋白基因的PRV为PRV614，通常将其单独或者与其他颜色PRV共同注射到小鼠的特定脑区，研究相关的神经环路。
在神经环路示踪研究中，通常需要同时使用2-3种不同荧光蛋白；在不同的脑区分别注射带有不同颜色荧光蛋白的PRV，经过一段时间后观察荧光蛋白的表达分布情况，被两种荧光同时标记到的神经元，说明同时与两个脑区有联系。例如，徐富强教授实验室曾向C57/BL小鼠两侧嗅球分别注射PRV152(GFP)和PRV614(RFP)，经过一段时间逆行，在嗅觉相关脑区观察到红色和绿色共同标记的神经元，说明这些神经元调控了双侧嗅球。
然而，目前已有的表达红色荧光蛋白的重组PRV病毒普遍存在荧光强度弱、成熟时间长、透射能力差等问题，不利于神经网络示踪研究。因此，有必要开发一种新的携带红色荧光蛋白的重组PRV，使其表达的红色荧光蛋白亮度更强、成熟速度更快，更好地应用于神经环路示踪。
发明内容
针对现有技术的不足及实际需求，本发明提供了一种表达E2-crimson的重组伪狂犬病毒及其制备方法和应用，所述重组伪狂犬病毒中采用的红色荧光蛋白E2-crimson荧光强度高、成熟时间快、透射能力强，在神经环路示踪领域具有广泛的应用前景。
为达此目的，本发明采用以下技术方案：
第一方面，本发明提供了一种重组伪狂犬病毒载体，所述重组伪狂犬病毒载体包括E2-crimson基因；
所述E2-crimson基因的核酸序列如SEQ ID NO:1所示；
SEQ ID NO:1所示的核酸序列为：
Figure PCTCN2020129494-appb-000001
本发明中，E2-Crimson是第一款采用标准远红光激光器有效激发的荧光蛋白，采用E2-crimson作为红色荧光蛋白，消光系数高达126000M -1cm -1，中等量子产率为0.23，亮度强，荧光强度是mRFP的2.3倍，成熟时间比mRFP快三倍，缩短了实验时间，E2-crimson在611nm处具有最大吸收，646nm处具有最大发射，相对于mRFP发生了明显的红移，透射能力强，在神经环路示踪领域具有显著的应用潜力。
优选地，所述重组伪狂犬病毒载体采用pBB5载体作为空载体，向pBB5 载体中连接入E2-crimson基因，得到重组伪狂犬病毒载体pBB5-E2-crimson。
本发明中，由于采用PRV152基因组作为重组亲本，因此在构造重组载体时，选择含有PRV152基因组同源重组臂的pBB5载体连接E2-crimson基因。
第二方面，本发明提供了一种如第一方面所述的重组伪狂犬病毒载体的制备方法，所述制备方法包括以下步骤：
采用限制性内切酶NsiI-HF处理pCMVGF-E2-crimson载体，同时采用限制性内切酶pstI-HF处理pBB5载体；
对两种酶切产物进行回收、连接，得到所述重组伪狂犬病毒载体pBB5-E2-crimson。
本发明中，限制性内切酶pstI-HF与NsiI-HF互为同尾酶，即两种内酶切酶切后产生的粘性末端可以互补配对，采用pstI-HF与NsiI-HF处理后的载体可以在连接酶作用下连接起来，形成新的重组载体，E2-crimson基因接到两个同源重组臂之间。
第三方面，本发明提供了一种重组伪狂犬病毒，所述重组伪狂犬病毒包括如第一方面所述的重组伪狂犬病毒载体。
第四方面，本发明提供了一种如第三方面所述的重组伪狂犬病毒的制备方法，所述制备方法包括以下步骤：
(1)采用限制性内切酶SpeI将重组伪狂犬病毒载体pBB5-E2-crimson进行线性化处理；
(2)提取PRV152病毒基因组；
(3)将线性化pBB5-E2-crimson与PRV152病毒基因组共转染宿主细胞，得到所述重组伪狂犬病毒。
根据本发明，步骤(1)采用限制性内切酶SpeI对重组载体pBB5-E2-crimson进行线性化处理，原因在于SpeI紧临5’同源重组臂，采用SpeI酶切重组载体后，5’和3’的同源重组臂以及中间的E2-crimson基因仍然连接在一起，没有被酶切分开，保证了下一步与PRV152基因组同源重组的顺利进行。
本发明中，采用PRV152病毒作为亲本，原因在于PRV152表达绿色荧光蛋白EGFP，而PRV-E2-crimson带红色荧光，利用荧光颜色的不同，方便在荧光显微镜下区分亲本PRV152和重组PRV-E2-crimson。
优选地，步骤(2)所述提取PRV152病毒基因组的方法包括：
采用PRV152病毒感染宿主细胞，使PRV152病毒在宿主细胞中扩增；收集宿主细胞，进行细胞裂解；DNA提取得到所述PRV152病毒基因组。
优选地，步骤(3)所述宿主细胞包括293细胞、293T细胞或293F细胞中的任意一种或至少两种的组合。
优选地，在步骤(3)之后还包括对重组伪狂犬病毒进行筛选和纯化的步骤。
本发明中，在绿色荧光PRV152病毒基础上，利用同源重组的方法，将E2-crimson基因连接在PRV152基因组上，构造出了能够稳定表达红色荧光蛋白E2-crimson、能够自我复制、具有逆向示踪功能的PRV-E2-crimson病毒，主要步骤如下：
(1)构建含有E2-crimson基因以及同源重组臂的重组载体；
(2)采用限制性内切酶线性化同源重组载体；
(3)提取含有相同同源重组臂的PRV152基因组；
(4)将线性化的同源重组载体与PRV152基因组共转染宿主细胞，通过同源重组制备得到含E2-crimson的重组PRV；
(5)多轮筛选，获得重组成功的表达红色荧光蛋白的PRV-E2-crimson；
(6)纯化PRV-E2-crimson病毒，注射到小鼠的纹状体中，检测PRV-E2-crimson的感染表达效率。
第五方面，本发明提供了一种神经网络示踪剂，所述神经网络示踪剂包括如第一方面所述的重组伪狂犬病毒载体和/或如第三方面所述的重组伪狂犬病毒。
第六方面，本方面提供了一种试剂盒，所述试剂盒包括如第一方面所述的重组伪狂犬病毒载体、如第三方面所述的重组伪狂犬病毒或如第五方面所述的神经网络示踪剂。
第七方面，本发明提供了一种如第一方面所述的重组伪狂犬病毒载体、如第三方面所述的重组伪狂犬病毒、如第五方面所述的神经网络示踪剂或如第六方面所述的试剂盒在制备神经系统疾病检测试剂中的应用。
与现有技术相比，本发明具有如下有益效果：
(1)本发明的重组PRV-E2-crimson采用E2-crimson作为红色荧光蛋白，荧光强度是mRFP的2.3倍，成熟时间比mRFP快三倍，缩短了实验时间，E2-crimson在611nm处具有最大吸收，646nm处具有最大发射，相对于mRFP发生了明显的红移，透射能力强；
(2)本发明的重组PRV-E2-crimson在小鼠纹状体中可以高效地逆向标记神经环路，表达效率高，侵染能力强；
(3)本发明的重组PRV-E2-crimson在神经环路示踪领域具有广泛的应用前景。
附图说明
图1为重组载体pBB5-E2-crimson的图谱和制备流程图；
图2(A)为PRV-E2-crimson进行第一轮筛选结果图，图2(B)为PRV-E2-crimson进行第二轮筛选结果图，图2(C)为PRV-E2-crimson进行第三轮筛选结果图；
图3为PRV-E2-crimson在小鼠纹状体及其上游环路的表达情况。
具体实施方式
为进一步阐述本发明所采取的技术手段及其效果，以下结合实施例和附图对本发明作进一步地说明。可以理解的是，此处所描述的具体实施方式仅仅用于解释本发明，而非对本发明的限定。
实施例中未注明具体技术或条件者，按照本领域内的文献所描述的技术或条件，或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可通过正规渠道商购获得的常规产品。
实施例1 构建重组载体
本实施例由于采用PRV152基因组作为重组亲本，因此在构造重组载体时，采用含有PRV152基因组同源重组臂的pBB5载体连接E2-crimson基因，步骤如下：
如图1所示，采用限制性内切酶pstI-HF(NEB)处理pBB5载体，同时采用限制性内切酶NsiI-HF(NEB)处理pCMVGF-E2-crimson载体，37℃下酶切3h，酶切体系如表1-1和表1-2所示，回收酶切产物后采用连接预混液(2×ligation premix，TAKARA)在16℃下进行连接过夜，体系如表1-3所示，得到连接成功的pBB5-E2-crimson载体。
表1-1 pBB5载体酶切体系
试剂 用量
酶切缓冲液10×CutSmart Buffer 3μL
pstI-HF 1μL
pBB5 1μg
ddH 2O 补齐至30μL
表1-2 pCMVGF-E2-crimson载体酶切体系
试剂 用量
酶切缓冲液10×CutSmart Buffer 3μL
NsiI-HF 1μL
pCMVGF-E2-crimson 1μg
ddH 2O 补齐至30μL
表1-3连接体系
试剂 用量
连接预混液2×ligation premix 5μL
pBB5连接载体 0.5μL
pCMVGF-E2-crimson连接片段 4.5μL
实施例2 重组载体的线性化处理
重组载体与PRV152进行同源重组前，进行线性化处理，步骤如下：
采用限制性内切酶SpeI对5μg重组载体pBB5-E2-crimson在37℃下进行线性化处理2h，体系如表2所示，酶切后采用酚氯仿抽提纯化DNA，得到线性化重组载体。
表2线性化酶切体系
试剂 用量
酶切缓冲液10×CutSmart Buffer 5μL
SpeI 2μL
pBB5-E2-crimson 5μg
ddH 2O 补齐至50μL
酚氯仿抽提纯化DNA的步骤如下：
向上述50μL线性化酶切体系中加入150μL ddH 2O，随后加入200μL酚氯仿抽提液，剧烈混匀；12000rpm 4℃离心10min，取上清约150μL置于新的1.5mL离心管中，加入20μL 3M醋酸钠溶液，加入500μL无水乙醇混匀，在-20℃冰箱放置半小时；12000rpm 4℃离心10min，弃上清，可以看到管底有白色沉淀，加入500μL无水乙醇洗涤DNA沉淀两次，最后一次洗涤后将上清倒掉，室温放置保证乙醇挥发完全，加入50μL ddH 2O溶解DNA沉淀，置于-20℃备用。
实施例3 PRV152基因组提取
本实施例采用PRV152病毒作为亲本，原因在于PRV152表达绿色荧光蛋白EGFP，而PRV-E2-crimson带红色荧光，利用荧光颜色的不同，方便在荧光显微镜下区分亲本PRV152和重组PRV-E2-crimson，PRV152基因组的提取步骤如下：
(1)采用PRV152病毒感染宿主细胞猪肾细胞PK15，使PRV152病毒在宿主细胞中扩增；
(2)收集PK15细胞，加入细胞裂解液，37℃水浴过夜；
(3)利用酚氯仿抽提法纯化PRV152基因组DNA，具体为在裂解液中加 入等体积的酚氯仿抽提液，上下剧烈摇晃混匀，4℃下12000rpm离心10min；吸取上清至新的EP管中，加入等体积的酚氯仿抽提液，剧烈摇晃混匀，4℃下12000rpm离心10min；重复上步一次；向上清中加入2倍体积的预冷的异丙醇，轻轻倒转混匀，4℃下5000rpm离心1min；重复上步一次；弃上清，沉淀采用800μL 75％乙醇洗涤，4℃下12000rpm离心5min；重复上步一次；弃上清，12000rpm离心5min，用枪头吸取残余液体，打开管盖，室温放置5min，加入50μL ddH 2O溶解DNA沉淀，置于-20℃中备用。
实施例4 重组PRV病毒的制备
本实施例利用钙转染的方法，将线性化pBB5-E2-crimson与PRV152基因组导入293T细胞中，两者在重组酶的作用下，借助同源重组臂，完成同源重组过程，得到重组PRV-E2-crimson。
提前一天将293T细胞铺于六孔板的一个孔中，保证第二天密度达到60％～70％；第二天，转染前4小时更换新鲜的完全培养基(含10％FBS+1％P/S)，转染试剂包括A(12.5μL2M CaCl 2，1μgPRV152基因组DNA，1μg线性化pBB5-E2-crimson DNA，2μg小牛胸腺DNA，用ddH 2O补齐至100μL)和B(100μL 2×HBS)；
A混合液配好后混合均匀，随后将B逐滴加入A，并用手轻弹混匀(或用涡旋仪混匀)；室温放置20min后，加入预先铺好的细胞中；
PRV152基因组DNA与线性化pBB5-E2-crimson拥有长同源臂和短同源臂，293T细胞中含有重组酶，两者在重组酶的作用下相互交换同源臂中的成分，完成同源重组。
实施例5 重组PRV病毒的筛选
由于同源重组效率有限，形成的病毒产物中，既有重组成功的红色荧光PRV-E2-crimson，也有绿色荧光亲本PRV152，本实施例通过筛选得到重组成功的PRV-E2-crimson，具体为：
将上清液进行不同浓度的稀释，加入到铺有密度为90％左右的PK15细胞的96孔板中，培养24h后在荧光显微镜下挑出只表达红色荧光不表达绿色荧光的孔，取上清继续重复上述筛选步骤，三轮后挑选出纯粹的红色荧光PRV-E2-crimson，如图2(A)、图2(B)和图2(C)所示，方框代表每一轮挑选出来的用作下一轮筛选的孔。
实施例6 小鼠纹状体中PRV-E2-crimson的感染表达效率
选取三只8周C57小鼠，向小鼠纹状体各注射2μL、滴度为2×10 9pfu/mL的PRV-E2-crimson，三天后小鼠出现明显的PRV感染迹象，行动迟缓、食欲不振、身体消瘦，将小鼠灌流取脑，利用免疫组化方法切片染色，观察脑片结果。
如图3所示，STR(striatum)为纹状体，是病毒的注射位点，Amy(amygdala)为杏仁核，STN(subthalamic nucleus)为丘脑底核，VTA(ventral tegmental area)为腹侧被盖区，可以看出，PRV-E2-crimson在纹状体及其上游环路有很高的表达效率。
综上所述，本发明的重组PRV-E2-crimson采用E2-crimson作为红色荧光蛋白，荧光强度是mRFP的2.3倍，成熟时间比mRFP快三倍，缩短了实验时间，对细菌和哺乳动物细胞无细胞毒性，E2-crimson在611nm处具有最大吸收，646nm处具有最大发射，相对于mRFP发生了明显的红移，透射能力强；所述重组PRV-E2-crimson在小鼠纹状体中可以高效地逆向标记神经环路，表达效率高，侵染能力强，在神经环路示踪领域具有广泛的应用前景。
申请人声明，本发明通过上述实施例来说明本发明的详细方法，但本发明并不局限于上述详细方法，即不意味着本发明必须依赖上述详细方法才能实施。所属技术领域的技术人员应该明了，对本发明的任何改进，对本发明产品各原料的等效替换及辅助成分的添加、具体方式的选择等，均落在本发明的保护范围和公开范围之内。

Claims (10)

  1. 一种重组伪狂犬病毒载体，其特征在于，所述重组伪狂犬病毒载体包括E2-crimson基因；
    所述E2-crimson基因的核酸序列如SEQ ID NO:1所示。
  2. 根据权利要求1所述的重组伪狂犬病毒载体，其特征在于，所述重组伪狂犬病毒载体的空载体为pBB5载体。
  3. 一种如权利要求1或2所述的重组伪狂犬病毒载体的制备方法，其特征在于，所述制备方法包括以下步骤：
    采用限制性内切酶NsiI-HF处理pCMVGF-E2-crimson载体，同时采用限制性内切酶pstI-HF处理pBB5载体；
    对两种酶切产物进行回收、连接，得到所述重组伪狂犬病毒载体pBB5-E2-crimson。
  4. 一种重组伪狂犬病毒，其特征在于，所述重组伪狂犬病毒包括如权利要求1或2所述的重组伪狂犬病毒载体。
  5. 一种如权利要求4所述的重组伪狂犬病毒的制备方法，其特征在于，所述制备方法包括以下步骤：
    (1)采用限制性内切酶SpeI将重组伪狂犬病毒载体pBB5-E2-crimson进行线性化处理；
    (2)提取PRV152病毒基因组；
    (3)将线性化pBB5-E2-crimson与PRV152病毒基因组共转染宿主细胞，得到所述重组伪狂犬病毒。
  6. 根据权利要求5所述的制备方法，其特征在于，步骤(2)所述提取PRV152病毒基因组的方法包括：
    采用PRV152病毒感染宿主细胞，使PRV152病毒在宿主细胞中扩增；收集宿主细胞，进行细胞裂解；DNA提取得到所述PRV152病毒基因组；
    优选地，步骤(3)所述宿主细胞包括293细胞、293T细胞或293F细胞中的任意一种或至少两种的组合。
  7. 根据权利要求5或6所述的制备方法，其特征在于，在步骤(3)之后还包括对重组伪狂犬病毒进行筛选和纯化的步骤。
  8. 一种神经网络示踪剂，其特征在于，所述神经网络示踪剂包括如权利要求1或2所述的重组伪狂犬病毒载体和/或如权利要求4所述的重组伪狂犬病毒。
  9. 一种试剂盒，其特征在于，所述试剂盒包括如权利要求1或2所述的重组伪狂犬病毒载体、如权利要求4所述的重组伪狂犬病毒或如权利要求8所述的神经网络示踪剂。
  10. 一种如权利要求1或2所述的重组伪狂犬病毒载体、如权利要求4所述的重组伪狂犬病毒、如权利要求8所述的神经网络示踪剂或如权利要求9所述的试剂盒在制备神经系统疾病检测试剂中的应用。
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