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无限制克隆技术研究进展及其应用

  • 杨传辉1,2

    机 构：

    1). 遵义医科大学珠海校区，珠海 51904

    2). 珠海市中药基础与应用研究重点实验室，珠海 519040

    ×
  • 卢荣锐1,2

    机 构：

    1). 遵义医科大学珠海校区，珠海 51904

    2). 珠海市中药基础与应用研究重点实验室，珠海 519040

    ×
  • 杨愈丰1,2

    机 构：

    1). 遵义医科大学珠海校区，珠海 51904

    2). 珠海市中药基础与应用研究重点实验室，珠海 519040

    角 色：通讯作者

    作者简介：

    ×

1. 遵义医科大学珠海校区，珠海 51904； 2. 珠海市中药基础与应用研究重点实验室，珠海 519040

中图分类号: Q81

DOI:10.16476/j.pibb.2019.0017

Research Progress and Application of Restriction Free Cloning Technology

  • YANG Chuan-Hui1,2

    Affiliation：

    1). Zhuhai Campus of Zunyi Medical University, Zhuhai 519040, Chin

    2). Zhuhai Key Laboratory of Fundamental and Applied Research in Traditional Chinese Medicine, Zhuhai Campus of Zunyi Medical University, Zhuhai 519040, China

    ×
  • LU Rong-Rui1,2

    Affiliation：

    1). Zhuhai Campus of Zunyi Medical University, Zhuhai 519040, Chin

    2). Zhuhai Key Laboratory of Fundamental and Applied Research in Traditional Chinese Medicine, Zhuhai Campus of Zunyi Medical University, Zhuhai 519040, China

    ×
  • YANG Yu-Feng1,2

    Affiliation：

    1). Zhuhai Campus of Zunyi Medical University, Zhuhai 519040, Chin

    2). Zhuhai Key Laboratory of Fundamental and Applied Research in Traditional Chinese Medicine, Zhuhai Campus of Zunyi Medical University, Zhuhai 519040, China

    Role：Corresponding author

    Profile：

    ×

1. Zhuhai Campus of Zunyi Medical University, Zhuhai 519040, Chin； 2. Zhuhai Key Laboratory of Fundamental and Applied Research in Traditional Chinese Medicine, Zhuhai Campus of Zunyi Medical University, Zhuhai 519040, China

CLC: Q81

DOI:10.16476/j.pibb.2019.0017

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目录 contents

    摘要

    无限制克隆（restriction-free cloning，RFC）是近年来建立起来的一种简单通用的DNA克隆技术，它可以精确地将目的片段插入到质粒内任意位置，是一种不受酶切位点、连接酶效率、目的基因长度或载体序列等条件限制的新型DNA重组技术．与其他多种克隆方法相比，RFC技术拥有不可替代的优势．本文在总结国内外RFC技术研究的基础上，系统阐述了RFC技术的原理、特点和在克隆方面的研究进展，并探讨其在分子生物学和合成生物学等领域的应用价值．

    Abstract

    Restriction free cloning(RFC) is a simple and universal DNA cloning technology developed in recent years, which can accurately insert the target DNA sequence into any position of any plasmids. It is a novel DNA recombination method that is not limited by restriction endonuclease, efficiency of ligase, length of target DNA sequence or vector sequence. Compared with other cloning methods, RFC technology has irreplaceable advantages. On the basis of summarizing the researches of RFC at home and abroad, this manuscript systematically introduces the action principle, characteristics and research progress of RFC technology and discuss its application value in molecular biology and synthetic biology.

    DNA克隆技术是指将目的基因片段插入到特定的质粒中形成重组质粒，进而实现目的基因的扩增、转录或翻译的一种基本分子生物学实验技术，在生物医学领域研究中该技术有着非常重要的应用[1]．传统的分子克隆方法由于采用酶切连接的方式，会受到限制性内切酶酶切位点和DNA连接酶连接效率等条件的限制，导致克隆成功率较低且耗时长、耗费高[2,3]．为高效地克隆目的基因[4]，近年来各研究领域的科研工作者根据自身需要，发展出了多种基于重组的新克隆方法．这些方法各有千秋，亦有相应的不足(表1)．其中以通用载体质粒融合系统(UPS)[5]等为代表的位点特异性重组克隆技术，主要适用于大规模基因的高通量克隆、表达载体及文库构建[6,7,8]．该方法在细菌、酵母、哺乳动物等不同宿主的载体中均可应用，通过借助重组酶及其特异识别位点，实现高效、靶向性强的基因克隆．然而由于位点特异性重组酶和辅助因子等的使用，导致实验成本较高．此外，由于特异识别位点的限制，该方法无法实现多个DNA片段的无缝克隆，亦不适用于涉及高复杂性或多片段克隆的合成生物学研究．而以辅助交配遗传整合型克隆法（MAGIC）[9] 等为代表的体内外同源重组克隆技术，则主要应用于文库的构建及多DNA片段组装[10,11,12,13,14]．该类方法既不需要使用位点特异性重组酶，也无需DNA的预处理，仅通过体内重组系统即可完成重组克隆．但MAGIC法需要准备供试菌株，基于细胞提取物的无缝连接克隆法（SLiCE）需要制备细胞提取物，基于PCR的IVA和OEPR克隆法可组装的片段长度和效率也会受到限制．再者，不依赖基因序列和连接反应克隆法（SLIC）[15,16,17]等基于单链退火拼接的克隆技术，主要应用于高通量克隆或多DNA片段组装的合成生物学研究[18,19,20,21,22,23,24,25,26,27]．这几类方法不受限制性酶切位点的限制，无需附加特定的重组位点，理论上均可实现无缝克隆．当然，其中一部分方法需要专利重组酶等，导致费用高昂，还有一部分方法需要对载体与目的基因进行预处理，产生单链同源突出末端，操作繁琐．更重要的是，其组装效率和正确率亦会随着DNA组装片段的增多而降低．

    尽管同样存在瑕疵，但无限制克隆（RFC）技术仍然值得关注了解[28,29,30]．RFC是Chen等[28]于2000年提出，而由Van Den Ent等[29]于2006年基于商业化定点突变技术改进而来的一种简单通用DNA克隆方法，同时也是一种可精确地将任何目的片段插入质粒中任意位置的分子克隆方法．并且近年来以RFC原理为基础，聚合酶环状延伸克隆（CPEC）[31,32]、依赖于体内重组的重叠延伸PCR克隆(OEPC)[33]、快步克隆(QuickStep-Cloning)[34]等多种克隆新技术相继被开发出来．RFC主要适用于构建重组载体、多DNA片段组装、定向突变、基因替换和高通量克隆．其应用范围广泛，是一种完全能够替代传统酶切连接的无缝克隆方法．该技术既无需限制性内切酶和连接酶，亦无需对载体进行预处理，也不依赖于特定的同源序列，打破了分子克隆的诸多限制，为分子克隆开辟一种全新的思路，使DNA重组技术更加方便实用．另外，在本论文审稿期间，Qaidi等[35]报道了一种基于重叠延伸PCR的克隆方式，属于广义的RFC技术．由于该技术尚未有充分的验证，本文不展开评述．

    表1 各种分子克隆方法的比较

    Table 1 Comparison of several molecular cloning methods

    原理克隆方法限制条件

    是否实现

    无缝克隆

    		适用范围文献
    酶切连接传统克隆方法存在酶切位点的限制否构建重组载体、定向突变、基因替换[2,3]

    位点特异性重组

    		UPS需要特异性重组位点、载体、重组酶否构建重组载体[5]
    Gateway需要特异性重组位点、载体、重组酶否构建重组载体、高通量克隆[6,7,8]

    体内同源重组

    		MAGIC需要特殊菌株是构建重组载体[9]

    IVA/RLF/ OEPR

    		线性化质粒和插入序列的比例需做适当优化

    是

    		构建重组载体、组装DNA大片段、定向突变、高通量克隆[10,11,12,14]

    体外同源重组

    SLiCE

    需要制备细胞提取物，操作繁琐

    是

    		构建重组载体、多DNA片段组装、高通量克隆

    [13]

    单链退火拼接

    SLIC

    需要T4 DNA聚合酶

    是

    		构建重组载体、组装中等DNA片段、高通量克隆

    [15,16,17]

    UDG需要尿嘧啶-DNA 糖基化酶否构建重组载体、高通量克隆[18,19,20]

    Gibson组装

    操作复杂，费用高，需T5核酸外切

    酶、DNA聚合酶及连接酶协同作用

    是

    构建重组载体、组装DNA大片段

    [21]

    In-Fusion/CloneEZ

    需要专利重组酶

    是

    		构建重组载体、多DNA片段组装、定向突变

    [22,23,24,25]

    TPA/AFEAP

    操作繁琐

    是

    		构建重组载体、多DNA片段组装、高通量克隆

    [26,27]

    聚合酶链式反应

    RFC/CPEC/OEPC/

    QuickStep-Cloning/ABC cloning

    依赖于扩增效率

    是

    构建重组载体、多DNA片段组装、定向突变、基因替换、高通量克隆

    [29,31,33,34,35,36,37,38]

  • 1　RFC技术的基本原理及特点

  • 1.1　RFC技术的基本原理

    RFC是基于位点定向突变技术演化而来的一种分子克隆技术[39,40]．与位点定向突变不同的是，RFC不仅可以实现碱基的突变、插入或删除，同时亦可引入完整的基因实现无缝克隆．RFC技术由一对长引物启动，该引物由目的基因与目标质粒插入位点两侧互补序列组成．之后以亲本质粒为模板，由高保真酶扩增后得到的PCR扩增产物经DpnⅠ降解除掉亲本质粒，最终经细菌的修复系统修复得到完整的重组质粒（图1）[33]．目前在保证较高效率构建重组质粒的情况下，该技术允许插入基因片段长度最长可达约15 kb[38]．

    图1 RFC技术的基本工作流程[41]

    图1 RFC技术的基本工作流程[41]

    Fig. 1 Basic work flow of RFC technology[41]

  • 1.2　RFC技术工作流程

    RFC的基本工作流程（图1）[41]：设计第一轮PCR引物（上下游引物的5'端均含约20～25 bp的目标质粒互补序列），以含有目的基因的序列为模板进行第一轮PCR扩增，产物作为一对长引物（包含目的基因序列及两侧目标质粒互补序列）用于二轮PCR反应．在第二轮PCR反应中，以从宿主分离得到的有甲基化的质粒为模板，待长引物退火结合到目标质粒上后，高保真酶通过PCR扩增目标质粒序列，从而将目的基因合并到一个有缺口的目标质粒DNA分子中．第二轮PCR产物经由DpnⅠ选择性降解甲基化的模板质粒，保留未甲基化的重组质粒，转化后阳性克隆通过测序确认．

  • 1.3　RFC技术的作用特点

    RFC是一种高效的 DNA 分子克隆新方法，并具有以下优势：a. RFC技术是一种序列非特异性克隆方法，无需选择特异性酶切位点，尤其适用于质粒上缺乏合适的限制性酶切位点情况下的分子克隆，使目的基因插入位点的选择自由度更高； b. RFC技术所需材料普通、操作简单、适用范围广，既不需要UDG或SLIC系统中的特殊的酶，亦不需要Gateway克隆中附加特定的重组序列即可完成质粒构建；c. 由于RFC本质上是两次PCR扩增，在现有高品质的商品化高保真酶高度普及的情况下，克隆过程出错率很低，最终获得的PCR产物经由DpnⅠ消化后便可降解模板质粒，因而大大地降低了假阳性的概率．总之，相比其他的克隆方法，该方法具有不需要加入不必要的序列、无需线性化载体、反应速率快、效率高和工作量小等优点，所以RFC技术是一种简单且实用性强的载体构建方法．

  • 2　RFC技术的应用

  • 2.1　在基因替换及改造中的应用

    当进行克隆或表达载体的构建时，目标质粒常常可能是含有其他目的基因的重组质粒．采用这种质粒为模板进行新的重组质粒构建前，必须切除原有的目的基因．此时倘若原有酶切位点不存在或不合适，重组质粒中原有的目的基因则不能去除，或无法用于新的重组质粒的构建．这种情况下，RFC技术恰好可以直接用新的目的基因替换掉原有的目的基因，一步到位完成克隆．同时，RFC还可以去除多余的侧翼序列．利用该方法，科研人员已实现了融合标签MBP和GB1之间的简便置换 （图2）[37]．此外，利用基因替换的功能，更可以顺利实现对基因的改造．并且在多位点突变的过程中，RFC技术展示了比定点定向诱变法[42,43]、多位点定向诱变法[44,45,46]等技术更明显的优势．该技术既可在不同指定位点同时引入多个位点特异性突变，又可在不同的位置分别插入或除去靶序列，同时还与AFEAP诱变法[47]一样拥有较高的效率和保真度．另外，以RFC为基础的转移PCR(TPCR)克隆通过使用多条包含突变碱基的上游引物和一条下游引物，可以实现多位点随机组合突变，构建多样化库[48]．

    图2 RFC在基因片段替换中的应用[37]

    图2 RFC在基因片段替换中的应用[37]

    Fig. 2 Application of RFC in gene fragment replacement[37]

  • 2.2　在构建共表达载体中的应用

    在大肠杆菌系统中，目标蛋白与其分子伴侣共表达往往能够帮助某些容易形成包涵体的蛋白质实现可溶表达，从而减少目标蛋白错误折叠及包涵体的形成[49,50]．但多组分共表达需要使用多个表达载体或采用具有多个启动子的载体[51,52]．将不同的目的基因克隆到多个表达载体时操作麻烦，而将不同的目的基因克隆到单个表达载体亦需要连续克隆．然而利用RFC技术不仅能够高效地构建出所需要的载体[53]，而且可同时将两个或多个基因克隆到同一个表达载体的不同位置．Unger等[37]利用RFC技术成功将鸟氨酸脱羧酶抗酶编码基因与鸟氨酸脱羧酶编码基因同时克隆至pACYC Duet中，共表达后形成鸟氨酸脱羧酶-抗酶复合物，参与多胺稳态的调控（图3）．

    图3 RFC技术在多基因片段重组中的应用[37]

    图3 RFC技术在多基因片段重组中的应用[37]

    Fig. 3 Application of RFC technology in multiple gene fragment recombination[37]

  • 2.3　在多组分组装中的应用

    虽然目前已有许多商业化载体可满足大部分普通DNA克隆和蛋白质表达的需要，但仍然有部分实验需要定制由多种组件组装的特殊载体．一般载体组装可以通过在引物中引入酶切位点，然后通过酶切连接将DNA组件组装成新载体，也可以借助依赖于体外同源重组的SLIC法[54]、AFEAP法[26]、TPA法[27]、OEPR法[12]或体内同源重组的IVA法[10]，在单个反应体系中实现多个DNA片段的组装．但以上方法需要提前对载体或其他DNA组件进行预处理，或依赖特殊的重组酶．RFC技术则允许直接在同一个反应体系中完成多个DNA组件组装并连接到目标载体，必要时还可以同时去除目标载体上多余的组件．Peleg等[36,37]利用RFC技术通过一个四通反应将链球菌标记序列、Trx融合标签和A32病毒粒子包装的ATPase基因进行组装，并连接到pET21质粒上，形成组装质粒pET21-Strep-Trx-L437，实现了L437的可溶性表达与纯化(图4)．

    图4 RFC在载体组装中的应用[36,37]

    图4 RFC在载体组装中的应用[36,37]

    Fig. 4 Application of RFC in vector assembly[36,37]

  • 2.4　在复杂基因文库构建中的应用

    随着生物科学的发展，新的克隆方法不断被建立和改良，以适应各种需求和应用．尤其是以创建复杂的组合合成基因库、基因电路和代谢途径等为重点的合成生物学的出现，需要更精确、高效、方便且经济的克隆技术．Gateway等[6]重组克隆技术由于需要目的基因和载体中特定的位点来构建重组质粒，通常不适合应用于涉及高复杂性或多片段克隆的合成生物学，因此不依赖于序列的RFC技术是构建复杂基因文库的首选[29]．Quan等[31,32]已论证了该技术在合成基因克隆、复杂组合文库和代谢途径等方面的广泛应用和优势．如图5所示，将两个库同时克隆到一个表达或功能筛选的载体中，以确定最佳组合序列．预计在合成生物学应用中，这种筛选将越来越频繁地进行，用于构建大分子复合物和识别最优的基因网络．

    图5 RFC在组合库克隆中的应用[32]

    图5 RFC在组合库克隆中的应用[32]

    Fig. 5 Application of RFC in combinatorial library cloning[32]

  • 2.5　在筛选蛋白质表达方案中的应用

    针对真核来源的蛋白质在原核系统中产物表达量低，或可溶表达情况较差等现象，以往通常利用Gateway克隆系统能够平行克隆重组蛋白的优势[55]，将靶蛋白基因并行克隆到不同表达系统进行最佳表达方案的筛选[56,57]．近年来发现，由于大部分表达载体组成相似，运用RFC技术优化蛋白质表达的同时更是提高了并行筛选蛋白质表达方案的灵活性(图6)．因此，RFC技术有可能替代Gateway技术成为蛋白质表达载体优化的最佳方案．

    图6 RFC在筛选表达方案中的应用

    图6 RFC在筛选表达方案中的应用

    Fig. 6 Application of RFC in screening expression scheme

  • 3　展 望

    自从重组DNA技术问世以来，作为现代分子生物学研究中基础且必需的工具，分子克隆技术被不断地更新和完善．随着合成生物学的迅速发展和普及，对分子克隆与组装技术的要求不断提高．作为近年来新开发的分子克隆技术，以PCR为基础的RFC技术摆脱了诸多的限制，真正意义上实现了无限制且精确地将目的片段克隆到任何载体上的任意位点．同时满足了分子生物学研究中日益增多的各种需要，成为传统酶切连接后的第二类通用型克隆方法．因而RFC技术作为一种分子生物学工具，在生物医学研究中展现出巨大的应用潜力．RFC技术凭借其强大的功能，可快速精确地将启动子和外显子等功能元件与侧翼序列的无缝连接，为分析生物体内基因功能提供了有力的技术支持．此外，RFC技术也为蛋白质工程或抗体工程中结构域的功能研究及合成生物学中新型超级蛋白质的设计提供了便利，保证结构域功能研究的准确性，提高不同功能结构域组合的效率．然而RFC虽兼具高效、通用、简单等优势，但其依赖于PCR的本质导致该技术对于一些富含G/C或重复序列的载体克隆效果较差．另外，如果目的基因片段过短，可能由于非特异性扩增或退火结合不牢固而易出现假阳性．因而如何提高PCR过程中引物结合的特异性与稳定性是RFC技术提高效率与增大阳性克隆率的关键．另外，可扩增的片段长度也受到高保真酶的限制而很难进一步突破．因此潜力巨大的RFC技术在具体的应用过程中同样需要进一步的优化，使得该技术在分子生物学研究方面应用更加广泛，进而为医学、生物学乃至合成生物学等领域的发展提供有力的技术支撑．

    Tel: 86-756-7637538, E-mail: yfyang@zmu.edu.cn

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  • 基本信息

    DOI:10.16476/j.pibb.2019.0017

    中图分类号: Q81

    引用信息

    杨传辉,卢荣锐,杨愈丰.无限制克隆技术研究进展及其应用[J].生物化学与生物物理进展,2019,46(07):719-726.

    YANG Chuan-Hui,LU Rong-Rui,YANG Yu-Feng.Research Progress and Application of Restriction Free Cloning Technology[J].Progress in Biochemistry and Biophysics,2019,46(07):719-726.

    基金信息

    国家自然科学基金（31560244），贵州省科技合作计划项目（黔科合LH字[2015]7547），遵义市“15851人才精英工程”科研项目和珠海市中药基础与应用研究重点实验室开放课题（ZYKL-2016K2）资助项目.

    This work was supported by grants from The National Natural Science Foundation of China(31560244), Science and Technology Cooperation Project of Guizhou Province(LH20157547), “15851 Talent Elite Project” of Zunyi City, Opening Project of Zhuhai Key Laboratory of Fundamental and Applied Research in Traditional Chinese Medicine(ZYKL-2016K2).

    稿件历史

    纸刊出版 : 2019-07-20
    收稿日期 : 2019-01-24
    录用日期 : 2019-05-06

    • 杨传辉

    机 构：

    1). 遵义医科大学珠海校区，珠海 51904

    2). 珠海市中药基础与应用研究重点实验室，珠海 519040

    Affiliation：

    1). Zhuhai Campus of Zunyi Medical University, Zhuhai 519040, Chin

    2). Zhuhai Key Laboratory of Fundamental and Applied Research in Traditional Chinese Medicine, Zhuhai Campus of Zunyi Medical University, Zhuhai 519040, China

    卢荣锐

    机 构：

    1). 遵义医科大学珠海校区，珠海 51904

    2). 珠海市中药基础与应用研究重点实验室，珠海 519040

    Affiliation：

    1). Zhuhai Campus of Zunyi Medical University, Zhuhai 519040, Chin

    2). Zhuhai Key Laboratory of Fundamental and Applied Research in Traditional Chinese Medicine, Zhuhai Campus of Zunyi Medical University, Zhuhai 519040, China

    杨愈丰

    机 构：

    1). 遵义医科大学珠海校区，珠海 51904

    2). 珠海市中药基础与应用研究重点实验室，珠海 519040

    Affiliation：

    1). Zhuhai Campus of Zunyi Medical University, Zhuhai 519040, Chin

    2). Zhuhai Key Laboratory of Fundamental and Applied Research in Traditional Chinese Medicine, Zhuhai Campus of Zunyi Medical University, Zhuhai 519040, China

    角 色：通讯作者

    Role：Corresponding author

    作者简介：

    Profile：

    原理克隆方法限制条件

    是否实现

    无缝克隆

    		适用范围文献
    酶切连接传统克隆方法存在酶切位点的限制否构建重组载体、定向突变、基因替换[2,3]

    位点特异性重组

    		UPS需要特异性重组位点、载体、重组酶否构建重组载体[5]
    Gateway需要特异性重组位点、载体、重组酶否构建重组载体、高通量克隆[6,7,8]

    体内同源重组

    		MAGIC需要特殊菌株是构建重组载体[9]

    IVA/RLF/ OEPR

    		线性化质粒和插入序列的比例需做适当优化

    是

    		构建重组载体、组装DNA大片段、定向突变、高通量克隆[10,11,12,14]

    体外同源重组

    SLiCE

    需要制备细胞提取物，操作繁琐

    是

    		构建重组载体、多DNA片段组装、高通量克隆

    [13]

    单链退火拼接

    SLIC

    需要T4 DNA聚合酶

    是

    		构建重组载体、组装中等DNA片段、高通量克隆

    [15,16,17]

    UDG需要尿嘧啶-DNA 糖基化酶否构建重组载体、高通量克隆[18,19,20]

    Gibson组装

    操作复杂，费用高，需T5核酸外切

    酶、DNA聚合酶及连接酶协同作用

    是

    构建重组载体、组装DNA大片段

    [21]

    In-Fusion/CloneEZ

    需要专利重组酶

    是

    		构建重组载体、多DNA片段组装、定向突变

    [22,23,24,25]

    TPA/AFEAP

    操作繁琐

    是

    		构建重组载体、多DNA片段组装、高通量克隆

    [26,27]

    聚合酶链式反应

    RFC/CPEC/OEPC/

    QuickStep-Cloning/ABC cloning

    依赖于扩增效率

    是

    构建重组载体、多DNA片段组装、定向突变、基因替换、高通量克隆

    [29,31,33,34,35,36,37,38]

    html/pibbcn/20190017/alternativeImage/b77fb8bd-806f-43d6-bc29-3527cb681358-F001.png
    html/pibbcn/20190017/alternativeImage/b77fb8bd-806f-43d6-bc29-3527cb681358-F002.png
    html/pibbcn/20190017/alternativeImage/b77fb8bd-806f-43d6-bc29-3527cb681358-F003.png
    html/pibbcn/20190017/alternativeImage/b77fb8bd-806f-43d6-bc29-3527cb681358-F004.png
    html/pibbcn/20190017/alternativeImage/b77fb8bd-806f-43d6-bc29-3527cb681358-F005.png
    html/pibbcn/20190017/alternativeImage/b77fb8bd-806f-43d6-bc29-3527cb681358-F006.png

    表1 各种分子克隆方法的比较

    Table 1 Comparison of several molecular cloning methods

    图1 RFC技术的基本工作流程[41]

    Fig. 1 Basic work flow of RFC technology[41]

    图2 RFC在基因片段替换中的应用[37]

    Fig. 2 Application of RFC in gene fragment replacement[37]

    图3 RFC技术在多基因片段重组中的应用[37]

    Fig. 3 Application of RFC technology in multiple gene fragment recombination[37]

    图4 RFC在载体组装中的应用[36,37]

    Fig. 4 Application of RFC in vector assembly[36,37]

    图5 RFC在组合库克隆中的应用[32]

    Fig. 5 Application of RFC in combinatorial library cloning[32]

    图6 RFC在筛选表达方案中的应用

    Fig. 6 Application of RFC in screening expression scheme

    image /

    无注解

    无注解

    无注解

    无注解

    无注解

    无注解

    无注解

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