抗胃癌单抗3H11可变区氨基端序列对抗体活性的影响

采用RT-PCR方法,利用第一骨架区通用引物扩增重链Fd段和κ链的基因,克隆到Fab表达载体中,在大肠杆菌中获得了表达但未检测到抗原结合活性.根据已克隆的3H11 VL、VH的真实序列,重新设计κ链及Fd段5′端引物,分别将骨架区引物在κ链及Fd段5′端所造成的氨基酸残基改变纠正为原始序列,构建分别含有矫正后κ链或矫正后Fd以及二个链均得到矫正的Fab表达载体,这些载体在大肠杆菌中均获得类似水平的表达,对任何一个链的矫正均可部分恢复Fab段胃癌细胞的结合活性.结果说明在构建小分子抗体时,PCR引物引入的轻、重链可变区氨基端氨基酸残基的改变可严重影响所表达抗体的抗原结合活性.     

Antibody Immunodiversity: A Study on the Marked Specificity Difference Between Two Anti-Yeast Iso-1 Cytochrome c Monoclonal Antibodies Whose Epitopes Are Closely Related

Anti-yeast iso-1 cytochrome c (cyt. c) monoclonal antibodies 2-96-12 and 4-74-6 have closely related epitopes (antigenic determinants). However, while the specificity of 4-74-6 is stringent, 2-96-12 cross-reacts with many evolutionarily related cytochromes c. Such a marked difference in specificity of antibodies with overlapping epitopes may represent unique antibody immunodiversity. Thus, we constructed Fv fragment models consisting of the variable domains of the heavy and light chains of 2-96-12 and 4-74-6 and that of another anti-iso-1 cyt. c as a control to gain insight into the origin of this difference in specificity. Our models show that 4-74-6 and 2-96-12 contain five and two aromatic side chains, respectively, in or near the central area of the antigen-combining site. The side chains of Arg95H (heavy chain) in 2-96-12 and Arg91L (light chain) in 4-74-6 project toward the central area of the combining site in our model. Antigen docking to our Fv models, combined with previous immunological studies, suggests that iso-1 cyt. c Asp60 may interact with Arg95H in 2-96-12 and Arg91L in 4-74-6 and that both epitopes of 2-96-12 and 4-74-7 may include iso-1 cyt. c Leu58, Asp60, Asn62, and Asn63. The effect of the Arg95H to Lys mutation on the antigen binding is also in accord with our model. The difference in specificity may be partly explained by a greater degree of conformational flexibility in and around the central area of the combining site in 2-96-12 compared to 4-74-6 due to differences in aromatic side chain packing.     

Development of a high yielding E. coli periplasmic expression system for the production of humanized Fab' fragments

Humanized Fab′ fragments may be produced in the periplasm of Escherichia coli but can be subject to degradation by host cell proteases. In order to increase Fab′ yield and reduce proteolysis we developed periplasmic protease deficient strains of E. coli. These strains lacked the protease activity of Tsp, protease III and DegP. High cell density fermentations indicated Tsp deficient strains increased productivity two fold but this increase was accompanied by premature cell lysis soon after the induction of Fab′ expression. To overcome the reduction in cell viability we introduced suppressor mutations into the spr gene. The mutations partially restored the wild type phenotype of the cells. Furthermore, we coexpressed a range of periplasmic chaperone proteins with the Fab′, DsbC had the most significant impact, increasing humanized Fab′ production during high cell density fermentation. When DsbC coexpression was combined with a Tsp deficient spr strain we observed an increase in yield and essentially restored “wild type” cell viability. We achieved a final periplasmic yield of over 2.4g/L (final cell density OD₆₀₀ 105), 40 h post Fab′ induction with minimal cell lysis.The data suggests that proteolysis, periplasm integrity, protein folding and disulphide bond formation are all potential limiting steps in the production of Fab′ fragments in the periplasm of E. coli. In this body of work, we have addressed these limiting steps by utilizing stabilized protease deficient strains and chaperone coexpression. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:212–220, 2017     

Efficient bacterial production of functional antibody fragments using a phagemid vector

The so-called ‘in vitro evolutionary method’ using a phage display system has been applied for protein engineering of the antigen-binding fragment of antibodies (Fab) by conducting random mutagenesis at the antigen-binding site in combination with antigen-based biopanning. However, isolated phage clones displaying Fab cannot necessarily be used for efficient bacterial production of engineered Fab proteins, often due to deleterious defects in their proper folding abilities derived in compensation for the gain of high affinity for a particular antigen. We here report a new method of an efficient and direct bacterial expression system for the phagemid-coded Fab proteins without use of the helper phage. To overcome a low folding efficiency derived from somatic hypermutations, if any, we have established optimum conditions for bacterial cultivation and protein expression, utilizing unusually long cultivation time (>50 h) and very low temperature (25 °C) and thereby leading to the production and extracellular secretion of Fab proteins in a very high yield (3–15 mg/L of culture). The purified Fab folded correctly and could efficiently bind an antigen, as judged by circular dichroism and isothermal titration calorimetry, respectively.     

Grain aphid population structure: no effect of fungal infections in a 2-year field study in Denmark

1 Sitobion avenae (F.) is a serious pest in Danish cereal crops. To understand the population genetic structure, aphids were sampled in seven different winter wheat (Triticum sativum Lamarck) fields throughout Denmark. The aphids were genotyped with seven microsatellite markers. In total, 2075 aphids were collected and 1203 of these were genotyped. 2 The Danish S. avenae populations displayed very high genotypic diversity, high percentages of unique genotypes and low linkage disequilibria; this is likely to be a result of genetic recombination encompassed by their holocyclic lifestyle. The populations showed very limited differentiation and no sign of isolation by distance. Almost all the genetic variation was ascribed within the populations rather than between populations, probably due to a high migration rate at approximate 10% per generation. 3 Seasonal changes in clonal diversity and distribution of asexual summer generations of S. avenae within the infestation period in a single winter wheat field were followed over two consecutive years by weekly sampling from 60 plots each of 20 × 20 m. Clonal diversity was high in all samples with no dominant clonal lineages and no significant difference in the genotypic diversity between weeks or between years. However, a temporal genetic differentiation effect, throughout the infestation, suggests that selective factors or high temporal migration play an important role in shaping the genetic structure S. avenae. 4 Analyses of fungal infected and uninfected aphids were performed to test whether some clonal linage were more often infected by fungi from the Entomophthorales under field conditions. In total, 54 progeny from aphids with Entomophthorales were genotyped and compared with 422 uninfected aphid genotypes. The Entomophthorales‐infected aphid genotypes did not cluster out together, suggesting that these fungal pathogens did not affect the population differentiation or clonal distribution of S. avenae in a Danish agroecosystem. 5 Our findings indicate that S. avenae populations can be controlled using conservation biological control     

麦长管蚜抗吡虫啉品系和敏感品系的生殖力比较

通过建立抗性品系和敏感品系的种群生命表,比较麦长管蚜Sitobion avenae(Fabricius)抗吡虫啉品系和敏感品系的生殖力差异,结果表明,抗性品系适合度显著下降,世代历期延长,成蚜寿命缩短;在繁殖上表现为产仔量下降,生殖期缩短。以净生殖率(R0)和内禀增长率(rm)来评价抗性品系的相对生物适合度,抗性品系分别是敏感品系的0.5200和0.7481。因此,麦长管蚜对吡虫啉的抗性品系存在显著的生殖劣势。     

Effects of Aphid Prey on Larval Development and Mortality of Adalia bipunctata and Coccinella septempunctata (Coleoptera: Coccinellidae)

Developmental time and mortality rate of Adalia bipunctata (L.) and Coccinella septempunctata L. (Coleoptera: Coccinellidae) were determined when feeding on five aphid species. Metopolophium dirhodum (Walker), Sitobion avenae (F.), Rhopalosiphum padi (L.), Hyalopterus pruni (Geoffr.) and Myzus cerasi F. (Homoptera: Aphididae) are widespread in Tekirda?, Turkey. Tests were carried out in a controlled environmental chamber (25±1°C temperature, 65±5% relative humidity and 16 h light:8 h dark period). Developmental times for A. bipunctata and C. septempunctata larvae varied significantly depending on species of aphid prey (P<0.05). Development time (±S.E.) varied from 17.50±0.84 to 20.83±1.60 days for C. septempunctata and 16.7±0.76 to 20.7±1.03 days for A. bipunctata. Mortality of A. bipunctata (50%) and C. septempunctata (63%) were highest on H. pruni.     

我国小麦抗麦长管蚜(Sitobion avenae)研究概况

麦长管蚜是我国小麦的重要害虫之一。选育和种植抗虫品种是防治麦长管蚜的理想方法。本文就我国小麦抗麦长管蚜种质资源筛选、抗性机制和小麦品种对麦蚜种群动态影响等方面的研究进展进行了概括,同时指出今后应加强小麦抗麦长管蚜基因的研究。     

Snowdrop lectin (Galanthus nivalis agglutinin) in aphid honeydew negatively affects survival of a honeydew- consuming parasitoid

1 Insecticidal proteins can be excreted in the honeydew when sap-sucking insects feed on insect-resistant transgenic plants. Honeydew can be an important source of carbohydrates, thus potentially exposing a broad range of honeydew-feeding insects to transgene products.
2 Snowdrop lectin ( Galanthus nivalis agglutinin; GNA) dissolved in a 2 m sucrose solution had no antifeedant effect on female aphid parasitoids ( Aphidius ervi ) but had a direct negative effect on their longevity.
3 When feeding on honeydew from Rhopalosiphum padi feeding on a GNA-containing artificial diet, Aphidius ervi suffered a longevity reduction that was more pronounced than was to be expected based on the detected GNA concentration in the honeydew.
4 Analysis of carbohydrate and amino acid composition revealed that a change in honeydew composition caused by a GNA-effect on the aphids could be a possible explanation for the additional reduction in parasitoid longevity.
5 When comparing the effect of honeydew from Sitobion avenae and R. padi feeding on GNA-expressing or nontransformed wheat plants on A. ervi longevity, aphid species was found to have a significant effect, whereas the wheat variety had no effect. The latter result was probably due to low GNA expression levels in the plants. Differences in nutritional suitability between honeydew from R. padi and S. avenae could be explained by differences in carbohydrate and amino acid composition.
6 This is the first study to demonstrate that GNA ingested by aphids and transported into the honeydew can negatively affect the parasitoids consuming this honeydew.
7 We recommend that honeydew should be considered as a route of exposure to transgene products in future risk assessment studies.