【作者】 郭小兵；

【导师】 张钦宪；

【作者基本信息】 郑州大学 ， 病理学与病理生理学， 2010， 博士

【摘要】 广谱抗菌药物,尤其是第三、四代头孢菌素的应用,为临床抗感染治疗作出贡献的同时,也逐步使其面临着严峻的考验。抗菌药物的不合理使用,对病原菌可形成选择压力并筛选出多重耐药菌株；多种基因转移元件的存在及其菌间频繁传递,可致多重耐药性的传播与流行。临床抗感染治疗时常面对无药可用的境地。探究菌株多重耐药机制,涉及产生水解酶与钝化酶、主动外排、药物作用靶位改变以及胞膜渗透性改变等,而最为关键的是产生了超广谱β-内酰胺酶。自1983年首例产超广谱β-内酰胺酶菌株发现以来,产酶菌株呈世界范围内流行。菌株种类涵盖肠杆菌科、非发酵菌科细菌等；单菌株携带酶型存在地域性分布特征；各型别酶抗药活性间存在一定差异。明确各地区超广谱β-内酰胺酶流行型别抗药活性,早期完成产酶菌株分离、鉴定,对于正确指导临床抗感染治疗,最终降低产酶菌株的产生几率及阻断产酶菌株流行意义重大。抗菌药物应用习惯差异,造成各地域间超广谱β-内酰胺酶流行型别不同。在我国大部分地区临床分离菌株所产酶型中,CTX-M型超广谱β-内酰胺酶占有相当比例。该型酶由德国学者Bauemfeind首先发现(CTX-M-1),目前已有数十种亚型,其分布地域及宿主菌谱极为广泛。CTX-M型酶水解底物为青霉素类、第一、二代头孢菌素,部分型别可水解头孢吡肟。基于酶蛋白结构构象特征以及药物自身空间位阻差异,CTX-M型酶对头孢噻肟的水解能力显著高于头孢他啶,部分菌株体外试验对头孢他啶显示高度敏感。根据氨基酸序列同源性分析结果,可将CTX-M型超广谱β-内酰胺酶分为5组,各组间氨基酸序列同源性小于90%,组内性大于94%。各组间DNA突变位点可位于编码基因的任一部位。进一步研究表明,尽管CTX-M型酶各亚型间核苷酸序列差异较大,但从酶蛋白结构上看,各突变位点多远离酶活性中心,对于酶蛋白抗药活性影响不大。故各亚型酶均具有CTX-M型酶的共有特性。基于此,明确一种型别CTX-M型酶的耐药谱,对于其他型别抗药活性的抑制具有指导价值。目前,临床实验室对于产酶菌株的鉴定主要采用三种方式,采用双纸片协同试验初步筛选阳性菌株；采用酶抑制剂增强试验/E-test试验进行确证；采用分子生物学试验进行快速特异性鉴定。前两者均需完成菌株的分离培养,耗时较长,难以满足临床简单快速的要求。后者试验费用较高,且实验软、硬件要求高,一般实验室难以开展。为了解决上述问题,作者采用双纸片协同及酶抑制剂增强试验筛选、收集46株产超广谱β-内酰胺酶大肠埃希菌；采用PCR技术克隆CTX-M型酶表达基因；采用基因重组技术构建CTX-M表达载体并表达；采用液体稀释法完成抗药活性检测。同时,通过免疫小鼠获得超广谱β-内酰胺酶特异性抗体；设计完善酶联免疫吸附试验,建立CTX-M型超广谱β-内酰胺酶的快速鉴定方法。通过上述试验,在明确CTX-M型酶抗药活性的同时,可获得其多克隆抗体,用于快速鉴定,乃至于被动免疫治疗。本研究包括以下三个部分。第一部分CTX-M-38型超广谱p-内酰胺酶的表达、定位及其抗药活性研究方法1.采用双纸片协同试验及酶抑制剂增强试验,对46株临床分离多重耐药大肠埃希菌进行超广谱β-内酰胺酶筛选与确证。2.依据CTX-M-38型超广谱β-内酰胺酶核苷酸序列特征,采用DNAstar分析软件,完成特异性PCR扩增引物设计。3.采用PCR技术,完成CTX-M-38型超广谱β-内酰胺酶全长编码基因的克隆。4.采用基因重组技术,完成pET-28a-CTX-M-38表达质粒的构建。5.采用氯化钙转化法,完成pET-28a-CTX-M-38表达质粒在BL21大肠埃希菌中的转化。6.采用卡那霉素抗性试验,检测BL21-pET-28a-CTX-M-38转化子构建效果。7．采用双脱氧链终止法对质粒插入片段进行核苷酸序列分析,检测所克隆CTX-M-38型编码基因序列突变状况。8.采用液体稀释法,检测BL21-pET-28a-CTX-M-38转化子抗药活性。9.采用K-B法药物敏感试验,检测培养液上清及菌体裂解液中CTX-M-38型超广谱β-内酰胺酶活性,初步进行酶蛋白定位研究。结果1.阿莫西林表现耐药,阿莫西林/克拉维酸表现敏感,两者交界处有协同现象,抑菌环直径明显扩大,产超广谱β-内酰胺酶菌株筛选试验阳性。2.头孢他啶抑菌环直径为8mm,头孢他啶/克拉维酸抑菌环直径为26mm,两者之差显著大于5mm,表明酶抑制剂增强试验阳性,产超广谱β-内酰胺酶菌株确证试验阳性。3.琼脂糖凝胶电泳显示,PCR扩增阳性产物大小与设计结果相一致。4.以转化子携带质粒为模板进行PCR扩增,约900bp位置处有目的条带,提示重组质粒构建成功。5.转化子在含卡那霉素液体培养基中生长良好,表明转化子构建成功。6.目的基因核苷酸序列与Bla CTX-M-38基因序列比对结果显示,两者在663位点处存在突变(T→A),但氨基酸序列上,两者221位均为丙氨酸,提示此处突变为同义突变。7.K-B法药敏试验中,滴加上清及菌体裂解液的头孢噻肟纸片均显示耐药,但后者抗药活性较为显著,单纯头孢噻肟纸片显示敏感,提示表达产物主要分布于菌体中。8.转化子对青霉素类药物包括哌拉西林、氨苄西林显著耐药(MIC>32μg/ml),对于头孢唑啉、头孢吡肟、头孢呋辛及头孢克洛MIC值>16μg/ml,表现为耐药。头孢噻肟及头孢曲松酶水解活性较强(MIC值均>32μg/ml)。头孢他啶MIC值为8μg/ml,表现为体外敏感。转化子对亚胺培南稳定敏感,MIC≤4μg/ml。酶抑制剂复合制剂中,哌拉西林/三唑巴坦敏感(MIC≤16/4μg/ml),而阿莫西林/棒酸、氨苄西林/舒巴坦均表现耐药(MIC>32/16μg/ml)。头孢哌酮／舒巴坦MIC为32/16μg/ml,表现为中介。氨曲南表现为敏感(MIC≤8μg/ml)。非内酰胺类抗菌药物中,庆大霉素、四环素、环丙沙星、左氧氟沙星及美满霉素均表现为耐药。第二部分CTX-M-38型超广谱β-内酰胺酶多克隆抗体的制备方法1.采用IPTG诱导BL21-pET-28a-CTX-M-38转化子表达酶蛋白。2.采用水煮裂解法,完成全菌蛋白提取。3.采用超声破碎法,完成包涵体蛋白提取。4.采用SDS-PAGE蛋白电泳技术,检测BL21-pET-28a-CTX-M-38转化子表达产物。5.采用扩散洗脱法,回收与纯化CTX-M-38型超广谱β-内酰胺酶蛋白。6.采用传统多克隆抗体制备方法,制备CTX-M-38型超广谱β-内酰胺酶蛋白特异性抗体。7.采用间接酶联免疫吸附试验,检测所制备多克隆抗体效价。8.采用辛酸沉淀法,对所制备的多克隆抗体进行纯化。9.采用Western blot印迹杂交技术,进行多克隆抗体的特异性鉴定。结果1. SDS-PAGE蛋白电泳结果显示,BL21-pET-28a-CTX-M-38转化子培养液上清、全菌蛋白、包涵体蛋白泳道,在约30kDa处出现明显蛋白条带,目的酶蛋白分子量大小与设计相同,表明在IPTG诱导作用下,转化子可显著表达酶蛋白。2. SDS-PAGE蛋白电泳后,各泳道酶蛋白量多少依次为包涵体蛋白、全菌蛋白以及培养液上清,提示转化子表达产物主要存在于包涵体内。3.间接酶联免疫吸附试验结果显示,多克隆抗体1：1000至1：16000梯度稀释度检测结果均呈阳性。1：32000以后稀释度时无阳性结果出现,表明多克隆抗体效价为1：16000,也表明多克隆抗体制备成功。4. Western blot印迹杂交技术检测结果显示,不同稀释度的抗体均与同一蛋白条带(约30kDa)反应,此条带与CTX-M-38型超广谱β-内酰胺酶蛋白大小相一致。提示研究中所制备多克隆抗体特异性较好。第三部分CTX-M-38型超广谱β-内酰胺酶多克隆抗体的应用研究方法1.采用梅花形双向琼脂扩散试验,完成CTX-M-38型超广谱β-内酰胺酶蛋白与多克隆抗体反应最适比检测。其中选择4h培养物作为试验抗原浓度,系列抗体滴度采用倍比稀释法制备。2.采用K-B法药物敏感试验,进行多克隆抗体抑制酶蛋白活性研究。3.采用改良过碘酸钠法,进行酶标多克隆抗体的制备。4.采用50%饱和硫酸铵沉淀法,进行酶标多克隆抗体的纯化。5.依据双抗体夹心法原理,设计制备酶联免疫吸附试验检测试剂盒。6.针对BL21-pET-28a-CTX-M-38转化子表达产物进行平行试验,检测批内变异,评价实验重复性。7.与PCR技术进行平行对照试验,完成临床分离产酶菌株检测,评价方法特异性。8.对27株产超广谱β-内酰胺酶大肠埃希菌(不携带CTX-M-38型超广谱β-内酰胺酶)进行检测,评价抗体交叉反应状况。9.对产CTX-M-38型超广谱β-内酰胺酶菌株不同时间段培养液进行检测,明确超广谱β-内酰胺酶最适检测时间。结果1.结果显示,抗体效价为1∶64时,沉淀线出现在两反应孔中间位置,表明抗体与4h培养物反应的最适效价为1∶64。2.K-B法药敏试验结果显示,抗体组抗菌药物抑菌环直径与对照组比较,p值小于0.01,两者结果有差异。表明CTX-M-38型超广谱β-内酰胺酶蛋白与多克隆抗体结合后,其活性受到一定程度的抑制。3.多克隆抗体抑制效应表现为阿莫西林、头孢他啶、头孢吡肟、氨曲南等药物的抑菌环直径增大,菌株由耐药转变为敏感或MIC进一步减小。含酶抑制剂复合药物两组结果基本相同。头孢噻肟抑菌环两组结果均无变化。4.对转化子表达产物30次平行试验结果显示,CV值为1.55%,提示本法批内变异小,具有较好的重复性。5.146株产超广谱β-内酰胺酶大肠埃希菌中,PCR技术检测阳性率为4.11%,本法为7.53%,x2检测可知P>0.05,表明两种检测方法检测结果无差异,提示本法具有一定的特异性。6.交叉反应试验中,27株临床分离产酶菌株中,有1株检测结果为阳性。表明所制备抗体具有交叉反应,实验结果存在假阳性现象。7.超广谱β-内酰胺酶最适检测时间结果显示,宿主菌在培养2h后,即有酶蛋白产生。4h时酶量显著增多,8h、18h及24h酶蛋白量已超过检测线性范围,故吸光度改变不大。结合检验流程,本研究确定超广谱β-内酰胺酶最适检测时间为菌株培养4h。结论1. CTX-M-38型超广谱β-内酰胺酶主要定位于宿主菌包涵体内；转化子具有广泛抗药活性,对部分抗菌药物抗药活性与其他型别存在差异。2.完成了CTX-M-38型超广谱β-内酰胺酶多克隆抗体的制备、纯化与鉴定,所制备多克隆抗体效价为1∶16000；抗体与CTX-M-38型超广谱β-内酰胺酶蛋白反应特异性较高。3.多克隆抗体可抑制酶蛋白抗药活性；双抗体夹心酶联免疫吸附试验检测酶蛋白具有较好的重复性、特异性；临床分离菌株产超广谱β-内酰胺酶最佳检测时间为菌株培养4h。更多还原

【Abstract】 With the usage of broad spectrum antibiotics, especially the third generation cephalosporins application, more infectious diseases were controlled and obtained better therapeutic efficacy. On the other hand, the overuse or abuse of antibiotics had made the clinic face a severe test gradually. Because selection pressure was formed, more and more multiple antibiotic resistant strains emerged recently. Many genetic transfer elements which contained resistant genes can transfer among the strains isolated from the clinic frequently, as result, the antibiotics resistance was spreaded at the same time. Without effective antibiotics, The clinic had no available project to treat the infectious disease sometimes. According to the results of latest research, we found that the antibiotic resistant mechanisms of the multiple antibiotic resistant strains involved in producing inactive and modification enzyme、changing membrane permeability and forming efflux pump. However, the most important things, especially for the Gram-negative bacterial, is producing extended-spectrumβ-lactamases (ESBLs).ESBL-producing strain was first reported in 1983. And nowdays, it has become an increasing problem in daily clinical life worldwide. The ESBLs-producing strains include Enterobacteriaceae, non-fermentative bacilli and so on; the ESBLs-types located in the single strain reveal diversity according to districts; different ESBLs type has different antibiotics resistance. So, it is very important for us to confirm the prevailing ESBLs types and its antibiotics resistance. And we must detect the ESBLs-producing strains early if we want to provide a correct treatment theory for the clinic.when all of these works are finished successfully, we can decrease the ESBLs-producing strains emerging rate and intercept the ESBLs-producing strains spreading indeed.Due to different antibacterials are used to treat infectious disease in different district, the prevalence of ESBL-types differs from country to country and from laboratory to laboratory.In most parts of china, the isolating rate of CTX-M type ESBLs was at a high level. The CTX-M-1 type ESBLs was first confirmed by Bauernfeind in German. Now, there were more subtypes were reported all over the world. The CTX-M type ESBLs may locate in more kinds of strains separated from different laboratory. The hydrolytic substrates of CTX-M type ESBLs include penicillins、the first and second generation cephalosporins. Some subtypes can hydrolyze cefecime. Because of the difference of stereospecific blockade between Cefotaxime and ceftazidime, the hydrolytic activity of enzyme to Cefotaxime is more stronger than to ceftazidime. And some strains producing CTX-M type ESBLs are noticeable sensitive in vitro. According to whole genome sequence nucleotide homology, the CTX-M type ESBLs can be divided into 5 clusters. The nucleotide homology is less than 90%among subtypes which belong to the different cluster, and more than 94%among subtypes which belong to in same group. The mutational sites among groups may locate at anywhere in the DNA sequences. The further researchs show that all of the DNA mutational sites are away from the enzyme active center, as result, all the CTX-M genotypes possess some common properties. Confirming the common properties is useful to inhibit the resistent activity of enzyme of CTX-M type ESBLs.Separating and identifying the ESBLs-producing strains in time is very important for the clinic to treat the infectious disease early and inhibit the resistent isolates spread effectively. Nowdays, there are three kinds of methods to identify the enzyme-producing strains in the clinical laboratory. The ESBLs-postive isolates are selected by double-disc synergy test and are confirmed by enzymes inhibitor enhancing test or E-test. Both of these methods can be performed base on the isolates separating and would consume a long time. The most available measure is the molecular biology technique which can detect the ESBLs-postive strains specificly and quickly. However, due to the high cost and the sophisticated laboratory requirement, molecular biology technique is inapplicable to be performed in clinical laboratory.In order to solve the problem discussed above,46 nonduplicate ESBLs-positive E.coli isolates were collected from the first affiliated hospital. ESBLs-Producing isolates were confirmed by double-disc synergy test and enzymes inhibitor enhancing test; The enzyme encoding genes were amplified by PCR; The construction and expression of the vecter of pET-28-CTX-M were preformed by gene recombination technique; The antibiotic resistence was detected by liquid dilution; we prepared and purified the polyclonal antibody through classical immunology methods; we established a quickly detective method of double antibody sandwich ELISA to confirm CTX-M type ESBLs. At last, we inhibited the enzyme abilities by Kirby-Bauer disc agar diffusion test to explore the application of the polyclonal antibody in seroimmunity.The experiment is divided into three parts. Part I:The analysis of expression and antibiotic susceptibilities of CTX-M-38 type extended-spectrum-lactamaseMethods:1.1.46 Multiple antibiotic resistant E.coli isolates were detected corresponding to the phenotype of producing ESBL detected by double-disc synergy screening test, and confirmation for producing ESBL was carried out by enzymes inhibitor enhancing test.2. Acorrding to nucleotide homology diversity of CTX-M-38 type ESBLs, specific primers were designed by DNAstar analysing software.3. CTX-M-38 type ESBLs encoding gene was amplified by PCR.4. The expressing vecter of pET-28-CTX-M-38 was constructed by gene recombination technique.5. Using CaCl2 transforming method to introduce the expressing vector of pET-28a-CTX-M-38 into E.coli BL21.6. Selecting the positive strains by kanamycin resistance and PCR.7. The insert element of pET-28-CTX-M-38 was detected by DNA sequencing to identified the mutation of CTX-M-38 type encoding gene.8. The antibiotic susceptibilities of the transformant of BL21-CTX-M-38 was carried out by liquid dilution test.9. The enzyme activities of culture supernatant and bacteria sonicate were tested by Kirby-Bauer disc agar diffusion to reflect its distribution.Results:1. The isolates was resistant to Amoxicillin and sensitive to Amoxicillin/clavulanic acid. There was synergy phenomenon in the juncture. All of these indicated that the screening test was positive. 2. The diameter of Ceftazidime was 8mm, and the diameter of Ceftazidime/ clavulanic acid was 26mm, the difference between them was larger than 5mm. All of these indicated that the confirmation test was positive.3. According to the agarose gel electrophoresis, the molecular weight of amplifying products corresponded to the design.4. Using PCR to detect the plasmids derived from transformants, the products located at 900bp in the agarose gel electrophoresis. The result indicated that the expressing vectors were constructed successfully.5. the transformant of BL21-CTX-M-38 could grow in the LB liquid medium (100 u g/ml kanamycin), the result indicated that the transformant of BL21-CTX-M-38 was constructed successfully.6. The mutation point in DNA sequence between insert element and Bla CTX-M-38 located at 663, the thymine was replaced by adenine. According to the analysis of amino acid sequence, however, the mutation was samesense mutation, the codon 221 of them all was alanine.7. According to the Kirby-Bauer disc agar diffusion test, the enzyme activities from bacteria sonicate was stronger than culture supernatant. The result showed that the product mainly lied inside the bacteria.8. The CTX-M-38 transformant was obviously resisitant to ampicillin and piperacillin, and their MICs were more than 32μg/ml. The transformant was noticeable resistant to the first、second and third generation cephalosporins tested in the study too. The MICs of cephazoline、cefuroxime、cefepime and cefaclor were all more than 16μg/ml, and the MIC of ceftriaxone was 32μg/ml. At the same time, the hydrolytic activity of enzyme to Cefotaxime was 4 times as much as it to the ceftazidime, the former was resistant and its MIC was more than 32 u g/ml, and the latter was sensitive in vitro and its MIC was 8μg/ml. On the other hand, The CTX-M-38 transformant was stably sensitive to imipenemwas and its MIC was less than 4μg/ml. Different from other CTX-M type ESBLs, the MIC of aztreonam was less than 8μg/ml, the result indicated that the transformant was sensitive to aztreonam. The transformant was sensitive to piperacillin/ tazobactam and its MIC was less than 16/4μg/ml. To the other antibiotics including beta-lactamase inhibitors including Amoxicillin/clavulanic and ampicillin/sulbactam, however, the transformant was resistance and their MIC were more than 32/16μg/ml. The MIC of cefoperazone/sulbactam was equal to 32/16μg/ml, which indicated that the transformant was intermediate to the antibiotic. The transformant was also resistance to gentamicin, minocycline, ciprofloxacin and levofloxacin.Part II:the polyclonal antibody preparation of CTX-M-38 type ESBLsMethods:1. The CX-M-38 transformant expressed the extended-spectrum beta-lactamases in BL21 E.coli with IPTG derivation.2. The bacterial protein was extracted by water boiling extraction technique.3. The inclusion body protein was extracted by sonication method.4. The expressing products of CTX-M-38 transformant was detected by SDS-PAGE electrophoresis technique.5. The CTX-M-38 type ESBLs was reclaimed and purified by elution diffusion method.6. The polyclonal antibody of CTX-M-38 type ESBLs was preparation by classical immunology methods. 7. The antibody titer of polyclonal antibody was detected by indirect enzyme-linked immunosorbent assay.8. The polyclonal antibody prepared in research was purified by Octanoic acid precipitation method.9. The specificity of the polyclonal antibody was confirmed by Western blot.Results:1. The SDS-PAGE electrophoresis results showed that there was a noticeable protein band in the supernatant, whole bacterial proteins and inclusion body protein lane respectively, and the molecular weight was about 30kDa, which correspond to the design.All of these indicated that the transformants could express enzyme protein significantly with IPTG induction.2. The SDS-PAGE electrophoresis results showed that the expressing products quantity was supernatant, whole bacterial proteins and inclusion body protein in turn. All these results indicated that the expressing products mainly lie in the inclusion body.3. According to the results of indirect enzyme-linked immunosorbent assay, we found that the experimental result was positive when the polyclonal antibody was diluted from 1:1000 to 1:16000, and the experimental result was negative when the antibody was diluted more than 1:32000. we could conclude that the antibody titer was 1:16000 and the polyclonal antibody was prepared successfully.4. The Western blot results showed that different dilutions of antibodies reacted to the same protein band (about 30kDa), which molecular weight was correspond to the CTX-M-38 type ESBLs. All of these results suggested that the specificity of polyclonal antibodies was better. PartⅢ:Study on the application of polyclonal antibody of CTX-M-38 type ESBLsMethods:1. The reaction optimal ratio between CTX-M-38 type ESBLs and polyclonal antibody was confirmed by double agar diffusion test. Selectinig 4h culture medium as the experimental antigen concentration,Series of antibody titers were prepared by fold dilution method.2. The Kirby-Bauer disc agar diffusion was carried out to explore the Inhibitory effect of enzyme activity by polyclonal antibody.3. The enzyme labelled antibody was prepared by modified heptaiodic acid sodium method.4. The enzyme labelled antibody was purified by 50%Saturated ammonium sulfate precipitation method.5. Based on double-antibody sandwich principle, we designed a quick detection method of enzyme-linked immunosorbent assay for CTX-M-38 type ESBLs.6. The parallel testing was carried out to detect the products of CTX-M-38 transformant. According the result, we obtained the information of experimental repeatability and intraasay variation.7.146 ESBLs-producing E.coli strains were detected by PCR and double-antibody sandwich ELISA respectively.Comparing with the results of the two groups, we evaluated the experimental specificity.8. Using double-antibody sandwich ELISA to detect 27 isolates of ESBL-producing E.coli strains(not carrying CTX-M-38 type ESBLs). According to the result, we evaluated the experimental cross-reactivity.9. The culture medium of CTX-M-38 type ESBLs-producing E.coli was detected at various time, Aaccording to the result, we confirmed the optimal detective time of ESBLs.Result:1. According to the double agar diffusion test result, we found that the Precipitation line located at the middle of two reactions when the antibody titer was 1:64. all of the results indicated that the optimal experimental ratio was 1:64.2. The antibiotic diameter of antibody group was different from the control group(p<0.01) in K-B disc agar diffusion, the result indicated that the polyclonal antibody could inhibit the enzyme activity to some degrees.3. The inhibition effect of polyclonal antibody to amoxicillin, ceftazidime, cefepime and aztreonam was obvious. The diameter of these four antibiotics of antibody group turned larger than that of control group, which indicated that the CTX-M-38 transformant turn sensitive or the MIC of them decreased to some extent. It looked like that the anbibody had no effect on the cefotaxime and antibiotics including beta-lactamase inhibitors, the diameters of them were similar between the antibody group and control group.4. According the results of the parallel testing, the CV was 1.55%, which indicated that the intraasay variation was small and the experimental repeatability was better.5. There were 6 positive isolates detected by PCR among 146 strains, and 11 isolates of double-antibody sandwich ELISA. According to the statistics,p>0.05, we could conclude that there was no difference between the two groups and double-antibody sandwich ELISA had better Specificity.6. There was 1 positive strains detected by double-antibody sandwich ELISA among 27 strains, which indicated that the antibody possessed cross-reactivity and the method may have the phenomenon of false-positive. 7. The enzyme protein could be confirmed after the host bacteria was cultured 2h, and the result became significant after 4h. The amount of enzyme protein exceed the linear range of detection when cultured 8h,18h and 24h.As result, there was no change in absorbance. Combination of test procedures, this study was to determine the optimal detection time for ESBLs was culture 4h.Conclusion:1. The expressing products mainly lies inside the inclusion body, The transformant is obviously resistant to most of antibiotics.And there are some differences between the CTX-M-38 and other ESBLs types.2. The polyclonal antibody of CTX-M-38 type ESBLs is prepared, purified and identified. The polyclonal titer is 1:16000, the reaction between the antibody and enzyme protein possess higher specificity.3. Polyclonal antibody can inhibit the enzyme activity to some extent. Double-antibody sandwich enzyme-linked immunosorbent assay establish in research possesse better repeatability and specificity. The optimal detection time for ESBLs is culture for 4h.更多还原