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学者姓名：林海英
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Abstract ：
1,3-丁二醇(1,3-butanediol)是应用于化工合成、食品药品、日化生产等领域的重要二元醇,以大肠杆菌BL21(DE3)为宿主菌,利用乙酰辅酶A乙酰基转移酶PhaA、NAD(P)H依赖型乙酰乙酰辅酶A还原酶PhaB1、丁醛脱氢酶Bld构建1,3-丁二醇代谢合成途径,在此基础上探究醛酮还原酶PA1127及NAD激酶(NAD kinase,NADK)对重组菌株代谢生产1,3-丁二醇的影响,并对产1,3-丁二醇重组菌株进行培养条件优化,以期提高重组菌株的1,3-丁二醇产量.采用多质粒共转化系统和多顺反子系统两种形式构建重组质粒,转化获得多酶共表达重组菌株.在转化试验或发酵结束后,利用丹磺酰法(DNS法)检测葡萄糖消耗量、通过吸光值检测比较菌体量和NADPH水平、采用GC和HPLC对1,3-丁二醇和副产物进行定量分析.成功构建由PhaA、PhaB1、Bld组成的1,3-丁二醇代谢合成途径,并实现葡萄糖向1,3-丁二醇的转化;NADK过表达使重组菌株合成1,3-丁二醇的能力提高,同时使乙酸、乳酸、乙醇等副产物产量增加,发酵过程培养条件及培养基组分的优化可提高重组菌株1,3-丁二醇产量达0.531 g/L.此结论可为1,3-丁二醇生物合成研究提供理论依据.
Keyword ：
1 1 3-丁二醇 3-丁二醇 NAD激酶 NAD激酶 代谢途径 代谢途径 醛/酮还原酶PA1127 醛/酮还原酶PA1127
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| GB/T 7714 | 林海英 , 于艺洁 , 陈怡平 et al. 1,3-丁二醇合成细胞的构建及优化 [J]. | 中国生物工程杂志 , 2024 , 44 (11) : 30-38 . |
| MLA | 林海英 et al. "1,3-丁二醇合成细胞的构建及优化" . | 中国生物工程杂志 44 . 11 (2024) : 30-38 . |
| APA | 林海英 , 于艺洁 , 陈怡平 , 张曼 . 1,3-丁二醇合成细胞的构建及优化 . | 中国生物工程杂志 , 2024 , 44 (11) , 30-38 . |
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Abstract ：
Objective: Chemical coupling agent and polylysine (PL) were used to conjugate goat anti-rabbit IgG and horseradish peroxidase (HRP) to increase the number of HRP connections on antibodies to amplify the sensitivity of immune detection, and the sensitivity was detected and compared through immunodetection applications, which is of great significance for subsequent immune diagnosis and biological research. Methods: Tylthioacetate (SATA) and Traut’s were optimized for the polymer HRP-PL, the feed ratio of Succinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate (Sulfo-SMCC) was optimized with goat anti-rabbit IgG and HRP-PL, and the reaction ratio of the two was optimized. The conjugated IgG-PL-HRP and commercial secondary antibody were detected by immunoblot, ELISA and immunohistochemistry, respectively, and the amplification of conjugated IgG-PL-HRP was calculated. Results: When the molar ratio of PL to HRP was 1∶5, the molar ratio of HRP-PL to Traut’s was 1∶15, the molar ratio of goat anti-rabbit IgG to Sulfo-SMCC was 1∶30, the molar ratio of goat anti-rabbit IgG to HRP-PL was 1∶10, the reaction efficiency was high. In the immune spot test, the minimum detection limits of commercial secondary antibody and conjugate IgG-PL-HRP were 2.5 μg and 312.5 ng, respectively, and the maximum dilution times were 50 and 100 times, respectively. In ELISA experiment, the maximum dilution times were 5 000 and 20 000, times respectively. In the immunohistochemical experiment, the detection specificity and intensity of conjugated IgG-PL-HRP were higher than those of commercial secondary antibody. Conclusion: The primary amplification conjugate IgG-PL-HRP was successfully synthesized and its immune detection signal amplification was about 3 ~ 7 times that of the commercial secondary antibody. It is of great significance to the following immunodiagnosis and biological research. © 2023, China Biotechnology Press. All rights reserved.
Keyword ：
Antibody conjugate Antibody conjugate Horseradish peroxidase Horseradish peroxidase Polylysine Polylysine Signal amplification Signal amplification
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| GB/T 7714 | Shen, Y. , Yao, B.-Y. , Yang, R.-N. et al. Preparation and application of immunoassay signal amplification antibody conjugates [J]. | China Biotechnology , 2023 , 43 (1) : 42-49 . |
| MLA | Shen, Y. et al. "Preparation and application of immunoassay signal amplification antibody conjugates" . | China Biotechnology 43 . 1 (2023) : 42-49 . |
| APA | Shen, Y. , Yao, B.-Y. , Yang, R.-N. , Kang, W.-B. , Lin, H.-Y. . Preparation and application of immunoassay signal amplification antibody conjugates . | China Biotechnology , 2023 , 43 (1) , 42-49 . |
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目的:以聚赖氨酸(polylysine,PL)为骨架提高辣根过氧化物酶(horseradish peroxidase,HRP)与羊抗兔IgG的连接数量,比较几种化学偶联剂的偶联效果,通过免疫检测技术对其灵敏度进行检测和比较.方法:对HRP与PL、聚合物HRP-PL与N-琥珀酰-S-乙酰乙酸(N-succinimidyl-S-acetylthioacetate,SATA)和2-亚氨基硫烷(Traut's)两种试剂、羊抗兔IgG与琥珀酰亚胺基4-(N-马来酰亚胺甲基)环己烷-1-羧酸酯[succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate,Sulfo-SMCC]、活化后羊抗兔IgG及HRP-PL进行摩尔比的优化;对偶联物IgG-PL-HRP及商品化二抗分别进行斑点免疫印迹、ELISA和免疫组化,并计算偶联物IgG-PL-HRP的检测放大倍数.结果:当PL与HRP摩尔比为1∶5,HRP-PL与Traut's摩尔比为1∶15,羊抗兔IgG与Sulfo-SMCC摩尔比为1∶ 30,羊抗兔IgG与HRP-PL摩尔比为1:10时,反应效率较高;商品化二抗及偶联物IgG-PL-HRP在斑点免疫印迹实验中的最低检测限分别为2.5μg和312.5 ng,最大稀释倍数分别为50和100倍;在ELISA实验中的最大稀释倍数分别为5 000和20 000倍;在免疫组化实验中偶联物IgG-PL-HRP的检测特异性及强度均大于商品化二抗.结论:成功合成抗体偶联物IgG-PL-HRP,且免疫检测信号放大倍数为商品化二抗的3～7倍,这对后续免疫诊断及生物学的研究具有重要意义.
Keyword ：
信号放大 信号放大 抗体偶联物 抗体偶联物 聚赖氨酸 聚赖氨酸 辣根过氧化物酶 辣根过氧化物酶
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| GB/T 7714 | 沈妍 , 姚玢妍 , 杨若楠 et al. 免疫检测信号放大抗体偶联物的制备及应用 [J]. | 中国生物工程杂志 , 2023 , 43 (1) : 42-49 . |
| MLA | 沈妍 et al. "免疫检测信号放大抗体偶联物的制备及应用" . | 中国生物工程杂志 43 . 1 (2023) : 42-49 . |
| APA | 沈妍 , 姚玢妍 , 杨若楠 , 康文斌 , 林海英 . 免疫检测信号放大抗体偶联物的制备及应用 . | 中国生物工程杂志 , 2023 , 43 (1) , 42-49 . |
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融合了跨膜肽的抗氧化酶可进入细胞，保护细胞免受放射损伤。然而跨膜肽的跨膜能力没有靶向性，其也可把抗氧化酶带入肿瘤细胞进而保护肿瘤细胞，降低放疗的效果。为此，根据多数肿瘤细胞微环境中存在活性基质金属蛋白酶(matrix metalloproteinase,MMP)-2或MMP-9的特点，在细胞跨膜肽R9与人铜、锌超氧化物歧化酶(superoxide dismutase 1,SOD1)和谷胱甘肽S-转移酶(glutathione S-transferase,GST)之间融合MMP-2/9的底物肽X，设计了融合蛋白GST-SOD1-X-R9。该蛋白在肿瘤微环境中可因MMP-2/9酶切底物肽X而失去跨...
Keyword ：
MMP-2/9敏感性 MMP-2/9敏感性 可跨膜 可跨膜 大肠杆菌表达体系 大肠杆菌表达体系 抗氧化酶 抗氧化酶 稳定性 稳定性 纯化 纯化
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| GB/T 7714 | 何火聪 , 林丽香 , 李玲玲 et al. 基质金属蛋白酶-2/9敏感型可跨膜融合抗氧化酶的表达、纯化和表征（英文） [J]. | 生物工程学报 , 2022 , 38 (09) : 3515-3527 . |
| MLA | 何火聪 et al. "基质金属蛋白酶-2/9敏感型可跨膜融合抗氧化酶的表达、纯化和表征（英文）" . | 生物工程学报 38 . 09 (2022) : 3515-3527 . |
| APA | 何火聪 , 林丽香 , 李玲玲 , 吴伦巧 , 林海英 , 潘剑茹 . 基质金属蛋白酶-2/9敏感型可跨膜融合抗氧化酶的表达、纯化和表征（英文） . | 生物工程学报 , 2022 , 38 (09) , 3515-3527 . |
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融合了跨膜肽的抗氧化酶可进入细胞,保护细胞免受放射损伤.然而跨膜肽的跨膜能力没有靶向性,其也可把抗氧化酶带入肿瘤细胞进而保护肿瘤细胞,降低放疗的效果.为此,根据多数肿瘤细胞微环境中存在活性基质金属蛋白酶(matrix metalloproteinase,MMP)-2或MMP-9的特点,在细胞跨膜肽R9与人铜、锌超氧化物歧化酶(superoxide dismutase 1,SOD1)和谷胱甘肽S-转移酶(glutathione S-transferase,GST)之间融合 MMP-2/9的底物肽X,设计了融合蛋白GST-SOD1-X-R9.该蛋白在肿瘤微环境中可因MMP-2/9酶切底物肽X而失去跨膜肽,从而无法进入肿瘤细胞,进而只能进入正常细胞.全基因合成SOD1-X-R9序列,并将其插入原核表达载体pGEX-4T-1中,得到表达质粒,并实现了GST-SOD1-X-R9融合蛋白的可溶表达.GST-SOD1-X-R9经硫酸铵沉淀和GST亲和层析纯化,分子量约为47kDa,与理论值一致.纯化的融合蛋白的SOD活性和GST活性分别为2 954 U/mg和328 U/mgoGST-SOD1-X-R9的SOD活性或GST活性在生理条件下几乎没有变化.该融合蛋白在溶液中可被胶原酶Ⅳ部分水解.分别建立了2D和3D培养的HepG2细胞模型来检验肿瘤微环境中的MMP-2活力对该蛋白跨膜能力的影响.在2D培养模型中,HepG2的MMP-2活力极低,但在3D培养模型中,随着培养时间的增加,HepG2肿瘤球的体积变大,其胞外MMP-2活力也随之增强.GST-SOD1-X-R9在2D培养的HepG2细胞中具有和GST-SOD1-R9蛋白一样的跨膜效率,但在3D培养的HepG2细胞球中的跨膜能力大大降低.本研究为后续深入研究GST-SOD1-X-R9靶向防护正常细胞的氧化损伤效应奠定了基础.
Keyword ：
MMP-2/9敏感性 MMP-2/9敏感性 可跨膜 可跨膜 大肠杆菌表达体系 大肠杆菌表达体系 抗氧化酶 抗氧化酶 稳定性 稳定性 纯化 纯化
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| GB/T 7714 | 何火聪 , 林丽香 , 李玲玲 et al. 基质金属蛋白酶-2/9敏感型可跨膜融合抗氧化酶的表达、纯化和表征 [J]. | 生物工程学报 , 2022 , 38 (9) : 3515-3527 . |
| MLA | 何火聪 et al. "基质金属蛋白酶-2/9敏感型可跨膜融合抗氧化酶的表达、纯化和表征" . | 生物工程学报 38 . 9 (2022) : 3515-3527 . |
| APA | 何火聪 , 林丽香 , 李玲玲 , 吴伦巧 , 林海英 , 潘剑茹 . 基质金属蛋白酶-2/9敏感型可跨膜融合抗氧化酶的表达、纯化和表征 . | 生物工程学报 , 2022 , 38 (9) , 3515-3527 . |
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The trans-translation system is recognized as an excellent target for developing new drugs to rapidly sterilize Mycobacterium tuberculosis (TB) infection and significantly shorten TB treatment duration. As a vital component of the trans-translation system for rescuing stalled ribosomes, the SmpB protein from Mycobacterium tuberculosis (MtbSmpB, 1-160 a. a.) mediates tmRNA binding to stalled ribosomes through forming a complex with tmRNA. So far, few works have been conducted to prepare, characterize biophysical properties and determine three-dimensional structure for the full-length MtbSmpB protein. In the present work, we successfully expressed and purified the His-tagged full-length MtbSmpB protein in Escherichia coli with a yield of 26.9 mg from 1 L of Luria Bertani medium. We also obtained MtbSmpB with a yield of 18.5 mg from 1 L of M9 minimal medium. The MtbSmpB protein showed a single band in SDS-PAGE with a molecular weight of similar to 20 kDa consistent with the measurement from MALDI-TOF-mass spectrometry. The dynamic light scattering experiment indicated that MtbSmpB existed in a monomeric form. Moreover, both circular dichroism and nuclear magnetic resonance (NMR) experiments exhibited that MtbSmpB was well structured, suggesting that it could be feasible to determine its solution structure by NMR spectroscopy. NMR titration experiments showed that MtbSmpB specifically bound to tmRNA. This work lays the essential basis for further determining the solution structure and dynamics of the full-length MtbSmpB protein.
Keyword ：
MtbSmpB MtbSmpB NMR NMR Protein expression and purification Protein expression and purification Protein-tmRNA interaction Protein-tmRNA interaction Tuberculosis Tuberculosis
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| GB/T 7714 | Yang, Juanjuan , Liu, Yindi , Xu, Shuli et al. Expression, purification and characterization of the full-length SmpB protein from Mycobacterium tuberculosis [J]. | PROTEIN EXPRESSION AND PURIFICATION , 2018 , 151 : 9-17 . |
| MLA | Yang, Juanjuan et al. "Expression, purification and characterization of the full-length SmpB protein from Mycobacterium tuberculosis" . | PROTEIN EXPRESSION AND PURIFICATION 151 (2018) : 9-17 . |
| APA | Yang, Juanjuan , Liu, Yindi , Xu, Shuli , Lin, Haiying , Meng, Chun , Lin, Donghai . Expression, purification and characterization of the full-length SmpB protein from Mycobacterium tuberculosis . | PROTEIN EXPRESSION AND PURIFICATION , 2018 , 151 , 9-17 . |
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嵌合抗原受体T细胞免疫疗法(chimeric antigen receptor-T,CAR-T),是通过体外激活和扩增肿瘤特异或非特异性杀伤细胞达到抗肿瘤效果,在肿瘤免疫治疗方面具有良好的应用前景.本研究构建靶向EGFRⅢ(epidermal growth factor receptor variant Ⅲ)的嵌合抗原受体(CAR)的重组慢病毒表达载体,利用慢病毒感染并筛选能够稳定表达该嵌合抗原受体的Jurkat细胞系.通过EGFRvⅢ分子刺激、与U87MG细胞共培养的方式检测细胞系的活化状况.结果显示,成功构建了pCDH-EGFRvⅢscFv-CAR-copGFP-T2A-puro慢病毒表达重组质粒,并筛选出可稳定表达EGFRⅢ-CAR的Jurkat细胞系.CCK-8法检测显示,EGFRvⅢ分子刺激12h的Jurkat-CAR细胞增殖率约是对照组的1.36倍(P＜0.05);ELISA法检测显示,与U87MG细胞共孵育后,细胞上清中IL-2的浓度约是单独培养分泌在上清中IL-2的1.625倍(P＜0.01).以上结果表明,稳定表达CAR的iurkat细胞,可以靶向性识别EGFRvⅢ分子及EGFRvⅢ阳性的靶细胞,并引起IL-2细胞因子释放,为后续临床细胞免疫治疗提供了理论基础.
Keyword ：
EGFRvⅢ EGFRvⅢ Jurkat细胞 Jurkat细胞 嵌合抗原受体 嵌合抗原受体 慢病毒 慢病毒
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| GB/T 7714 | 高蕊 , 詹杜清 , 杜明明 et al. 肿瘤特异性嵌合抗原受体EGFRvⅢ-CAR的构建及体外活性分析 [J]. | 中国生物化学与分子生物学报 , 2018 , 34 (2) : 221-228 . |
| MLA | 高蕊 et al. "肿瘤特异性嵌合抗原受体EGFRvⅢ-CAR的构建及体外活性分析" . | 中国生物化学与分子生物学报 34 . 2 (2018) : 221-228 . |
| APA | 高蕊 , 詹杜清 , 杜明明 , 张芸 , 董欣 , 祝婧雯 et al. 肿瘤特异性嵌合抗原受体EGFRvⅢ-CAR的构建及体外活性分析 . | 中国生物化学与分子生物学报 , 2018 , 34 (2) , 221-228 . |
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The efforts were focused on exploring alternative pneumococcal vaccine strategies, aimed at addressing the shortcomings of existing formulations, without compromising efficacy. Our strategy involved the use of the carrier protein, pneumococcal surface protein A (PspA), conjugated with capsular polysaccharides (CPS), to provide effective and non-serotype-dependent protection. In this study, we generated a stable Escherichia coli construct expressing functional PspA from a capsular serotype 6B strain and confirmed it belonging to family I, which was conjugated with CPS. The distribution of anti-CPS antibody response was almost completely of IgG2a subclass followed by IgG3 and low level of IgG1 subclass, but that of anti-PspA IgG subclass antibodies was almost equal IgG1 and IgG2a subclasses. Though PspA was less conspicuous on the surface of pneumococci than the capsule, the antibodies induced with CPS-rPspA conjugate possessed more accessibility to the surface of Streptococcus pneumoniae serotype 6B and 19F (the same family 1 PspA). By survival experiment, the result suggested that the level of cross-protection after immunized with the conjugate was more measurable within the same family I. The CPS-rPspA conjugate not only induced CPS-specific protection but also provided PspA specific cross-protection. (c) 2015 Elsevier Ltd. All rights reserved.
Keyword ：
Capsular polysaccharides Capsular polysaccharides Conjugate Conjugate Family 1 Family 1 PspA PspA Streptococcus pneumoniae Streptococcus pneumoniae
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| GB/T 7714 | Lin, Haiying , Peng, Yonghui , Lin, ZiLin et al. Development of a conjugate vaccine against invasive pneumococcal disease based on capsular polysaccharides coupled with PspA/family 1 protein of Streptococcus pneumoniae [J]. | MICROBIAL PATHOGENESIS , 2015 , 83-84 : 35-40 . |
| MLA | Lin, Haiying et al. "Development of a conjugate vaccine against invasive pneumococcal disease based on capsular polysaccharides coupled with PspA/family 1 protein of Streptococcus pneumoniae" . | MICROBIAL PATHOGENESIS 83-84 (2015) : 35-40 . |
| APA | Lin, Haiying , Peng, Yonghui , Lin, ZiLin , Zhang, Shuangling , Guo, Yanghao . Development of a conjugate vaccine against invasive pneumococcal disease based on capsular polysaccharides coupled with PspA/family 1 protein of Streptococcus pneumoniae . | MICROBIAL PATHOGENESIS , 2015 , 83-84 , 35-40 . |
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目的构建肺炎链球菌家族Ⅰ中支系1和2肺炎链球菌表面蛋白A(PspA)重组融合蛋白,分析融合蛋白的免疫原性。方法 PCR扩增目的基因片段,构建重组质粒并表达,ELISA检测抗体滴度和亲和度,以及抗蛋白血清特异性和免疫交叉性。结果成功构建重组质粒并原核表达。血清中抗体滴度可达到1×10~4,亲和度达到2×10~5,调理吞噬试验阳性,保护试验效果显著。结论肺炎链球菌家族Ⅰ中支系1和2肺炎链球菌表面蛋白A(PspA)重组融合蛋白具有较高的免疫原性,能够诱导小鼠产生高滴度和高特异性的抗体,具有免疫交叉保护性。
Keyword ：
免疫原性 免疫原性 肺炎链球菌 肺炎链球菌 肺炎链球菌表面蛋白A 肺炎链球菌表面蛋白A 重组蛋白 重组蛋白
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| GB/T 7714 | 林海英 , 彭永辉 , 张双玲 et al. 肺炎链球菌表面蛋白A的家族Ⅰ不同支系融合蛋白的制备及免疫原性研究 [J]. | 中华微生物学和免疫学杂志 , 2015 , 35 (05) : 377-381 . |
| MLA | 林海英 et al. "肺炎链球菌表面蛋白A的家族Ⅰ不同支系融合蛋白的制备及免疫原性研究" . | 中华微生物学和免疫学杂志 35 . 05 (2015) : 377-381 . |
| APA | 林海英 , 彭永辉 , 张双玲 , 罗春华 , 郑美云 , 吕唯 et al. 肺炎链球菌表面蛋白A的家族Ⅰ不同支系融合蛋白的制备及免疫原性研究 . | 中华微生物学和免疫学杂志 , 2015 , 35 (05) , 377-381 . |
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Abstract ：
采用生物信息学方法分析几种病原性弧菌OmpK、OmpU和OmpW蛋白种间和种内的同源性,筛选高保守性蛋白OmpW.成功构建具备稳定分泌抗体IgG3的细胞株S5C10,鉴定其对5种弧菌具有特异性交叉免疫反应,而对9种非弧菌无免疫反应.研究结果显示OmpW可作为广谱性疫苗成分,其单抗可特异性检测多元弧菌.
Keyword ：
交叉免疫原性 交叉免疫原性 单抗 单抗 外膜蛋白 外膜蛋白 广谱性 广谱性 弧菌 弧菌
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| GB/T 7714 | 林海英 , 马振宁 , 郑允权 et al. 弧菌广谱性外膜蛋白抗原筛选及其单抗的交叉免疫原性研究 [J]. | 福州大学学报（自然科学版） , 2012 , 40 (4) : 535-540 . |
| MLA | 林海英 et al. "弧菌广谱性外膜蛋白抗原筛选及其单抗的交叉免疫原性研究" . | 福州大学学报（自然科学版） 40 . 4 (2012) : 535-540 . |
| APA | 林海英 , 马振宁 , 郑允权 , 郭养浩 , 唐凤翔 . 弧菌广谱性外膜蛋白抗原筛选及其单抗的交叉免疫原性研究 . | 福州大学学报（自然科学版） , 2012 , 40 (4) , 535-540 . |
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