CN1566159A - 一种能特异杀死肿瘤细胞的基因工程重组蛋白 - Google Patents

一种能特异杀死肿瘤细胞的基因工程重组蛋白 Download PDF

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CN1566159A
CN1566159A CN 03137587 CN03137587A CN1566159A CN 1566159 A CN1566159 A CN 1566159A CN 03137587 CN03137587 CN 03137587 CN 03137587 A CN03137587 A CN 03137587A CN 1566159 A CN1566159 A CN 1566159A
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CN1294149C (zh
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陈瑞雪
卢娟
赵欣欣
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Beijing Northland Biotech Co Ltd
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李相哲
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Abstract

本专利涉及一种能特异杀死肿瘤细胞的蛋白质，经过基因工程改造以后，杀伤肿瘤细胞的活性大大增强，在肿瘤治疗方面有着很大药用潜力。

Description

一种能特异杀死肿瘤细胞的基因工程重组蛋白
一.技术领域
癌症是目前严重威胁人们健康的一种疾病，据报道，全球每年癌症新发病人约1000万，我国每年新发癌症患者近100万，死亡约150万。目前对癌症治疗并没有非常有效的方法，如果早期发现可以使用手术，但大部分是在后期发现，只能通过放疗或化疗来进行治疗。放疗法一般使用阿霉素等药物，具有很大的副作用，主要有骨髓抑制而造成的各种血相降低，胃肠道反应，脱发，肝功能损伤和神经系统损伤等。而放疗的副作用更大，给病人带来巨大的痛苦。并且放化疗的有效率也不高，在30％左右。
鉴于此，急需找到一种副作用小并且有效率高的方法来对癌症进行治疗，随着现代生物技术的不断发展，这种方法成为了可能：由于放化疗副作用大的一个十分重要的原因是其毒性缺乏选择性，其在杀伤癌症细胞的同时也大量地杀伤了正常细胞，而生物导向技术有望解决这个问题，选用一些导向分子，如特异表达受体的配体或者受体的特异性抗体等来作为导向，连接上毒素，即是生物靶向药物，可以靶向的到达肿瘤部位，选择性地杀伤肿瘤细胞，达到治疗肿瘤的同时毒性很小的目的。
二.背景技术
关于生物靶向性药物，目前的导向分子有人促黄体生成激素释放因子(LHRH)，表皮生长因子(EGF)和白细胞介素2等，其中LHRH仅有10个氨基酸组成，分子量小，融合后不会影响毒素蛋白的空间构象，关于毒素部分，可选的有假单胞菌外毒素A(PE)，白喉毒素，蓖麻毒素等，目前较常见的是假单胞菌外毒素A(PE)，PE是一个由613个氨基酸组成的蛋白分子，去掉N末端1-252位氨基酸后就成为了PE40分子，由于分子量的减少，PE40成为一个常用的毒素。目前有报道的生物靶向性药物有白细胞介素2-白喉毒素，表皮生长因子-白喉毒素和LHRH-PE40，特异抗体连接细胞毒素等，但是该类毒素的临床用量较大，导致副作用较大，所以截止目前为止，生物靶向性药物只有白细胞介素2-白喉毒素在美国上市，而其他种类的药物还处于临床或临床前阶段，本专利针对生物靶向性药物效价低、用量大的特点，研究了一系列的生物导向融合蛋白，其具有生物活性高，用量小的特点。
三.发明内容
本专利主要是研究LHRH-PE40系列融和蛋白的基因工程改造，从而提高其杀伤癌细胞的生物活性，我们主要从以下2方面进行改造：1.对融合蛋白的LHRH部分进行改造，增强其与癌细胞结合能力；2.对融合蛋白的细胞毒素PE40部分进行改造，增强其癌细胞杀伤能力；3同时对融合蛋白的LHRH和PE40两方面进行改造，增强其癌细胞杀伤能力，从而达到减少用药量，降低体内毒副反应，延缓抗体产生时间，降低生产成本，增强治疗效果的目的，具有广阔的临床效益和社会效益。
LHRH作为融和蛋白导向部分氨基酸序列为：
Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly
LHRH突变体氨基酸序列分别为：
Glu-His-Trp-Ser-Tyr-Ala-Leu-Arg-Pro-Gly
Glu-His-Trp-Ser-Tyr-Trp-Leu-Arg-Pro-Gly
Glu-His-Trp-Ser-Tyr-Leu-Leu-Arg-Pro-Gly
假单胞杆菌外毒素A(PE40)作为融和蛋白毒素部分氨基酸序列为
253-279
Gly-Gly-Ser-Leu-Ala-Ala-Leu-Thr-Ala-His-Gln-Ala-Cys-His-Leu-Pro-Leu-Glu-Thr-Phe-Thr-Arg-
His-Arg-Gln-Pro-Arg
280-300
Gly-Trp-Glu-Gln-Leu-Glu-Gln-Cys-Gly-Tyr-Pro-Val-Gln-Arg-Leu-Val-Ala-Leu-Tyr-Leu-Ala
301-330
Ala-Arg-Leu-Ser-Trp-Asn-Gln-Val-Asp-Gln-Val-Ile-Arg-Asn-Ala-Leu-Ala-Ser-Pro-Gly-Ser-Gly-
Gly-Asp-Leu-Gly-Glu-Ala-Ile-Arg
331-364
Glu-Gln-Pro-Glu-Gln-Ala-Arg-Leu-Ala-Leu-Thr-Leu-Ala-Ala-Ala-Glu-Ser-Glu-Arg-Phe-Val-Arg-
Gln-Gly-Thr-Gly-Asn-Asp-Glu-Ala-Gly-Ala-Ala-Asn
365-399
Ala-Asp-Val-Val-Ser-Leu-Thr-Cys-Pro-Val-Ala-Ala-Gly-Glu-Cys-Ala-Gly-Pro-Ala-Asp-Ser-Gly-
Asp-Ala-Leu-Leu-Glu-Arg-Asn-Tyr-Pro-Thr-Gly-Ala-Glu
400-613
Phe-Leu-Gly-Asp-Gly-Gly-Asp-Val-Ser-Phe-Ser-Thr-Arg-Gly-Thr-Gln-Asn-Trp-Thr-Val-Glu-Arg-
Leu-Leu-Gln-Ala-His-Arg-Gln-Leu-Glu-Glu-Arg-Gly-Tyr-Val-Phe-Val-Gly-Tyr-His-Gly-Thr-Phe-
Leu-Glu-Ala-Ala-Gln-Ser-Ile-Val-Phe-Gly-Gly-Val-Arg-Ala-Arg-Ser-Gln-Asp-Leu-Asp-Ala-Ile-
Trp-Arg-Gly-Phe-Tyr-Ile-Ala-Gly-Asp-Pro-Ala-Leu-Ala-Tyr-Gly-Tyr-Ala-Gln-Asp-Gln-Glu-Pro-
Asp-Ala-Arg-Gly-Arg-Ile-Arg-Asn-Gly-Ala-Leu-Leu-Arg-Val-Tyr-Val-Pro-Arg-Ser-Ser-Leu-Pro-
Gly-Phe-Tyr-Arg-Thr-Ser-Leu-Thr-Leu-Ala-Ala-Pro-Glu-Ala-Ala-Gly-Glu-Val-Glu-Arg-Leu-Ile-
Gly-His-Pro-Leu-Pro-Leu-Arg-Leu-Asp-Ala-Ile-Thr-Gly-Pro-Glu-Glu-Glu-Gly-Gly-Arg-Leu-Glu-
Thr-Ile-Leu-Gly-Trp-Pro-Leu-Ala-Glu-Arg-Thr-Val-Val-Ile-Pro-Ser-Ala-Ile-Pro-Thr-Asp-Pro-Arg-
Asn-Val-Gly-Gly-Asp-Leu-Asp-Pro-Ser-Ser-Ile-Pro-Asp-Lys-Glu-Gln-Ala-Ile-Ser-Ala-Leu-Pro-
Asp-Tyr-Ala-Ser-Gln-Pro-Gly-Lys-Pro-Pro-Arg-Glu-Asp-Leu-Lys
(一).首先对融合蛋白假单胞杆菌外毒素A部分进行改造：
1.本专利对假单胞杆菌外毒素A PE40(氨基酸253-613)进行缺失替换突变，缺失替换其C末端5个氨基酸即609-613位氨基酸REDLK置换为XYEL，其中X代表赖氨酸或精氨酸，Y代表天冬氨酸，谷氨酰胺或谷氨酸。E代表谷氨酸，L代表亮氨酸。
2.本专利对假单胞杆菌外毒素A PE40进行缺失突变，缺失其N末端氨基酸253-279及氨基酸365-380部分，取其氨基酸280-364和氨基酸381-613部分按原顺序首位相连得到蛋白即为PE35。
3.本专利对PE35 C末端5个氨基酸REDLK置换为XYEL，其中X代表赖氨酸或精氨酸，Y代表天冬氨酸，谷氨酰胺或谷氨酸。E代表谷氨酸，L代表亮氨酸。
4.本专利取假单胞杆菌外毒素A缺失氨基酸365-380部分，取其氨基酸253-364和氨基酸381-613部分按原顺序首位相连得到蛋白即为PE38。
5.本专利对PE38 C末端5个氨基酸REDLK置换为XYEL，其中X代表赖氨酸或精氨酸，Y代表天冬氨酸，谷氨酰胺或谷氨酸。E代表谷氨酸，L代表亮氨酸。
6.本专利取假单胞杆菌外毒素A缺失氨基酸253-279部分，取其氨基酸280-613部分得到蛋白即为PE37。
7.本专利对PE37 C末端5个氨基酸REDLK置换为XYEL，其中X代表赖氨酸或精氨酸，Y代表天冬氨酸，谷氨酰胺或谷氨酸。E代表谷氨酸，L代表亮氨酸。
(二).另外对融合蛋白的LHRH部分进行改造：
1.本专利将天然LHRH的N末端第一位的焦谷氨酸改为谷氨酸，然后将其与突变后的PE40进行融合连接，或者将突变了的LHRH与PE40进行融合连接，形成高亲和力融和蛋白。
2.本专利将一、中融和蛋白LHRH部分的N末端第6位突变为Ala或Trp或Leu。
(三).将LHRH或突变了的LHRH部分的N末端与毒素部分的C末端进行连接，并在LHRH或突变了的LHRH的C末端连接上XYEL或REDLK。
(四).将末尾含有XYEL或REDLK序列的融合蛋白末端序列XYEL或REDLK重复串联2-5次。
总结：对融合蛋白LHRH部分的突变使其对肿瘤细胞的亲和力增强；对融合蛋白毒素部分的改变使得融合蛋白的毒力增强；二个部分的同时改变使得融合蛋白的杀伤肿瘤细胞的活性更是大大增强。
四.具体实施方式
实例一
首先从cDNA文库中用PCR方法得到LHRH与PE40的基因片段。然后再用PCR与基因重组方法把融合蛋白PE40部分C末端5个氨基酸突变为KDEL，并获得重组基因载体pLHRH-PE40(KDEL)，对插入载体的基因片段LHRH-PE40(KDEL)进行序列测定。验证其基因序列的正确性后，扩增保存，从该载体上切取下目的基因片段，插入表达载体中，再把重组表达载体导入表达宿主菌中，经培养表达，疏水层析和离子交换层析得到目的蛋白LHRH-PE40(KDEL)，用MTT细胞测活法测定LHRH-PE40(KDEL)的杀伤肿瘤细胞的活力。
此序列为LHRH-PE40(KDEL)(序列1)
Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-Gly-Gly-Ser-Leu-Ala-Ala-Leu-Thr-Ala-His-Gln-Ala-
Cys-His-Leu-Pro-Leu-Glu-Thr-Phe-Thr-Arg-His-Arg-Gln-Pro-Arg-Gly-Trp-Glu-Gln-Leu-Glu-Gln
-Cys-Gly-Tyr-Pro-Val-Gln-Arg-Leu-Val-Ala-Leu-Tyr-Leu-Ala-Ala-Arg-Leu-Ser-Trp-Asn-Gln-Val-
Asp-Gln-Val-Ile-Arg-Asn-Ala-Leu-Ala-Ser-Pro-Gly-Ser-Gly-Gly-Asp-Leu-Gly-Glu-Ala-Ile-Arg-
Glu-Gln-Pro-Glu-Gln-Ala-Arg-Leu-Ala-Leu-Thr-Leu-Ala-Ala-Ala-Glu-Ser-Glu-Arg-Phe-Val-Arg-
Gln-Gly-Thr-Gly-Asn-Asp-Glu-Ala-Gly-Ala-Ala-Asn-Ala-Asp-Val-Val-Ser-Leu-Thr-Cys-Pro-Val-
Ala-Ala-Gly-Glu-Cys-Ala-Gly-Pro-Ala-Asp-Ser-Gly-Asp-Ala-Leu-Leu-Glu-Arg-Asn-Tyr-Pro-Thr
-Gly-Ala-Glu-Phe-Leu-Gly-Asp-Gly-Gly-Asp-Val-Ser-Phe-Ser-Thr-Arg-Gly-Thr-Gln-Asn-Trp-
Thr-Val-Glu-Arg-Leu-Leu-Gln-Ala-His-Arg-Gln-Leu-Glu-Glu-Arg-Gly-Tyr-Val-Phe-Val-Gly-Tyr-
His-Gly-Thr-Phe-Leu-Glu-Ala-Ala-Gln-Ser-Ile-Val-Phe-Gly-Gly-Val-Arg-Ala-Arg-Ser-Gln-Asp-
Leu-Asp-Ala-Ile-Trp-Arg-Gly-Phe-Tyr-Ile-Ala-Gly-Asp-Pro-Ala-Leu-Ala-Tyr-Gly-Tyr-Ala-Gln-
Asp-Gln-Glu-Pro-Asp-Ala-Arg-Gly-Arg-Ile-Arg-Asn-Gly-Ala-Leu-Leu-Arg-Val-Tyr-Val-Pro-Arg-
Ser-Ser-Leu-Pro-Gly-Phe-Tyr-Arg-Thr-Ser-Leu-Thr-Leu-Ala-Ala-Pro-Glu-Ala-Ala-Gly-Glu-Val-
Glu-Arg-Leu-Ile-Gly-His-Pro-Leu-Pro-Leu-Arg-Leu-Asp-Ala-Ile-Thr-Gly-Pro-Glu-Glu-Glu-Gly-
Gly-Arg-Leu-Glu-Thr-Ile-Leu-Gly-Trp-Pro-Leu-Ala-Glu-Arg-Thr-Val-Val-Ile-Pro-Ser-Ala-Ile-Pro-
Thr-Asp-Pro-Arg-Asn-Val-Gly-Gly-Asp-Leu-Asp-Pro-Ser-Ser-Ile-Pro-Asp-Lys-Glu-Gln-Ala-Ile-
Ser-Ala-Leu-Pro-Asp-Tyr-Ala-Ser-Gln-Pro-Gly-Lys-Pro-Pro-Lys-Asp-Glu-Leu
实例二
从pLHRH-PE40(KDEL)载体用PCR方法把融合蛋白PE40部分C末端4个氨基酸突变为RQEL，得到目的基因片段LHRH-PE40(RQEL)进行序列测定。验证其基因序列的正确性后，扩增保存，从该载体上切取下目的基因片段，插入表达载体中，再把重组表达载体导入表达宿主菌中，经培养表达，纯化得到目的蛋白LHRH-PE40(RQEL)，用MTT细胞测活法测定LHRH-PE40(RQEL)的杀伤肿瘤细胞的活力。
此序列为LHRH-PE40(RQEL)(序列2)
Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-Gly-Gly-Ser-Leu-Ala-Ala-Leu-Thr-Ala-His-Gln-Ala-
Cys-His-Leu-Pro-Leu-Glu-Thr-Phe-Thr-Arg-His-Arg-Gln-Pro-Arg-Gly-Trp-Glu-Gln-Leu-Glu-Gln
-Cys-Gly-Tyr-Pro-Val-Gln-Arg-Leu-Val-Ala-Leu-Tyr-Leu-Ala-Ala-Arg-Leu-Ser-Trp-Asn-Gln-Val-
Asp-Gln-Val-Ile-Arg-Asn-Ala-Leu-Ala-Ser-Pro-Gly-Ser-Gly-Gly-Asp-Leu-Gly-Glu-Ala-Ile-Arg-
Glu-Gln-Pro-Glu-Gln-Ala-Arg-Leu-Ala-Leu-Thr-Leu-Ala-Ala-Ala-Glu-Ser-Glu-Arg-Phe-Val-Arg-
Gln-Gly-Thr-Gly-Asn-Asp-Glu-Ala-Gly-Ala-Ala-Asn-Ala-Asp-Val-Val-Ser-Leu-Thr-Cys-Pro-Val-
Ala-Ala-Gly-Glu-Cys-Ala-Gly-Pro-Ala-Asp-Ser-Gly-Asp-Ala-Leu-Leu-Glu-Arg-Asn-Tyr-Pro-Thr
-Gly-Ala-Glu-Phe-Leu-Gly-Asp-Gly-Gly-Asp-Val-Ser-Phe-Ser-Thr-Arg-Gly-Thr-Gln-Asn-Trp-
Thr-Val-Glu-Arg-Leu-Leu-Gln-Ala-His-Arg-Gln-Leu-Glu-Glu-Arg-Gly-Tyr-Val-Phe-Val-Gly-Tyr-
His-Gly-Thr-Phe-Leu-Glu-Ala-Ala-Gln-Ser-Ile-Val-Phe-Gly-Gly-Val-Arg-Ala-Arg-Ser-Gln-Asp-
Leu-Asp-Ala-Ile-Trp-Arg-Gly-Phe-Tyr-Ile-Ala-Gly-Asp-Pro-Ala-Leu-Ala-Tyr-Gly-Tyr-Ala-Gln-
Asp-Gln-Glu-Pro-Asp-Ala-Arg-Gly-Arg-Ile-Arg-Asn-Gly-Ala-Leu-Leu-Arg-Val-Tyr-Val-Pro-Arg-
Ser-Ser-Leu-Pro-Gly-Phe-Tyr-Arg-Thr-Ser-Leu-Thr-Leu-Ala-Ala-Pro-Glu-Ala-Ala-Gly-Glu-Val-
Glu-Arg-Leu-Ile-Gly-His-Pro-Leu-Pro-Leu-Arg-Leu-Asp-Ala-Ile-Thr-Gly-Pro-Glu-Glu-Glu-Gly-
Gly-Arg-Leu-Glu-Thr-Ile-Leu-Gly-Trp-Pro-Leu-Ala-Glu-Arg-Thr-Val-Val-Ile-Pro-Ser-Ala-Ile-Pro-
Thr-Asp-Pro-Arg-Asn-Val-Gly-Gly-Asp-Leu-Asp-Pro-Ser-Ser-Ile-Pro-Asp-Lys-Glu-Gln-Ala-Ile-
Ser-Ala-Leu-Pro-Asp-Tyr-Ala-Ser-Gln-Pro-Gly-Lys-Pro-Pro-Arg-Gln-Glu-Leu
实例三
从pLHRH-PE40(RQEL)载体用PCR方法把融合蛋白PE40部分C末端4个氨基酸突变为KEEL，得到目的基因片段LHRH-PE40(KEEL)进行序列测定。验证其基因序列的正确性后，扩增保存，从该载体上切取下目的基因片段，插入表达载体中，再把重组表达载体导入表达宿主菌中，经培养表达，纯化得到目的蛋白LHRH-PE40(KEEL)，用MTT细胞测活法测定LHRH-PE40(KEEL)的杀伤肿瘤细胞的活力。
此序列为LHRH-PE40(KEEL)(序列3)
Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-Gly-Gly-Ser-Leu-Ala-Ala-Leu-Thr-Ala-His-Gln-Ala-
Cys-His-Leu-Pro-Leu-Glu-Thr-Phe-Thr-Arg-His-Arg-Gln-Pro-Arg-Gly-Trp-Glu-Gln-Leu-Glu-Gln
-Cys-Gly-Tyr-Pro-Val-Gln-Arg-Leu-Val-Ala-Leu-Tyr-Leu-Ala-Ala-Arg-Leu-Ser-Trp-Asn-Gln-Val-
Asp-Gln-Val-Ile-Arg-Asn-Ala-Leu-Ala-Ser-Pro-Gly-Ser-Gly-Gly-Asp-Leu-Gly-Glu-Ala-Ile-Arg-
Glu-Gln-Pro-Glu-Gln-Ala-Arg-Leu-Ala-Leu-Thr-Leu-Ala-Ala-Ala-Glu-Ser-Glu-Arg-Phe-Val-Arg-
Gln-Gly-Thr-Gly-Asn-Asp-Glu-Ala-Gly-Ala-Ala-Asn-Ala-Asp-Val-Val-Ser-Leu-Thr-Cys-Pro-Val-
Ala-Ala-Gly-Glu-Cys-Ala-Gly-Pro-Ala-Asp-Ser-Gly-Asp-Ala-Leu-Leu-Glu-Arg-Asn-Tyr-Pro-Thr
-Gly-Ala-Glu-Phe-Leu-Gly-Asp-Gly-Gly-Asp-Val-Ser-Phe-Ser-Thr-Arg-Gly-Thr-Gln-Asn-Trp-
Thr-Val-Glu-Arg-Leu-Leu-Gln-Ala-His-Arg-Gln-Leu-Glu-Glu-Arg-Gly-Tyr-Val-Phe-Val-Gly-Tyr-
His-Gly-Thr-Phe-Leu-Glu-Ala-Ala-Gln-Ser-Ile-Val-Phe-Gly-Gly-Val-Arg-Ala-Arg-Ser-Gln-Asp-
Leu-Asp-Ala-Ile-Trp-Arg-Gly-Phe-Tyr-Ile-Ala-Gly-Asp-Pro-Ala-Leu-Ala-Tyr-Gly-Tyr-Ala-Gln-
Asp-Gln-Glu-Pro-Asp-Ala-Arg-Gly-Arg-Ile-Arg-Asn-Gly-Ala-Leu-Leu-Arg-Val-Tyr-Val-Pro-Arg-
Ser-Ser-Leu-Pro-Gly-Phe-Tyr-Arg-Thr-Ser-Leu-Thr-Leu-Ala-Ala-Pro-Glu-Ala-Ala-Gly-Glu-Val-
Glu-Arg-Leu-Ile-Gly-His-Pro-Leu-Pro-Leu-Arg-Leu-Asp-Ala-Ile-Thr-Gly-Pro-Glu-Glu-Glu-Gly-
Gly-Arg-Leu-Glu-Thr-Ile-Leu-Gly-Trp-Pro-Leu-Ala-Glu-Arg-Thr-Val-Val-Ile-Pro-Ser-Ala-Ile-Pro-
Thr-Asp-Pro-Arg-Asn-Val-Gly-Gly-Asp-Leu-Asp-Pro-Ser-Ser-Ile-Pro-Asp-Lys-Glu-Gln-Ala-Ile-
Ser-Ala-Leu-Pro-Asp-Tyr-Ala-Ser-Gln-Pro-Gly-Lys-Pro-Pro-Lys-Glu-Glu-Leu
实例四
从pLHRH-PE40(KEEL)载体用PCR方法把融合蛋白PE40部分C末端4个氨基酸突变为REEL，得到目的基因片段LHRH-PE40(REEL)进行序列测定。验证其基因序列的正确性后，扩增保存，从该载体上切取下目的基因片段，插入表达载体中，再把重组表达载体导入表达宿主菌中，经培养表达，纯化得到目的蛋白LHRH-PE40(REEL)，用MTT细胞测活法测定LHRH-PE40(REEL)的杀伤肿瘤细胞的活力。
此序列为LHRH-PE40(REEL)(序列4)
Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-Gly-Gly-Ser-Leu-Ala-Ala-Leu-Thr-Ala-His-Gln-Ala-
Cys-His-Leu-Pro-Leu-Glu-Thr-Phe-Thr-Arg-His-Arg-Gln-Pro-Arg-Gly-Trp-Glu-Gln-Leu-Glu-Gln
-Cys-Gly-Tyr-Pro-Val-Gln-Arg-Leu-Val-Ala-Leu-Tyr-Leu-Ala-Ala-Arg-Leu-Ser-Trp-Asn-Gln-Val-
Asp-Gln-Val-Ile-Arg-Asn-Ala-Leu-Ala-Ser-Pro-Gly-Ser-Gly-Gly-Asp-Leu-Gly-Glu-Ala-Ile-Arg-
Glu-Gln-Pro-Glu-Gln-Ala-Arg-Leu-Ala-Leu-Thr-Leu-Ala-Ala-Ala-Glu-Ser-Glu-Arg-Phe-Val-Arg-
Gln-Gly-Thr-Gly-Asn-Asp-Glu-Ala-Gly-Ala-Ala-Asn-Ala-Asp-Val-Val-Ser-Leu-Thr-Cys-Pro-Val-
Ala-Ala-Gly-Glu-Cys-Ala-Gly-Pro-Ala-Asp-Ser-Gly-Asp-Ala-Leu-Leu-Glu-Arg-Asn-Tyr-Pro-Thr
-Gly-Ala-Glu-Phe-Leu-Gly-Asp-Gly-Gly-Asp-Val-Ser-Phe-Ser-Thr-Arg-Gly-Thr-Gln-Asn-Trp-
Thr-Val-Glu-Arg-Leu-Leu-Gln-Ala-His-Arg-Gln-Leu-Glu-Glu-Arg-Gly-Tyr-Val-Phe-Val-Gly-Tyr-
His-Gly-Thr-Phe-Leu-Glu-Ala-Ala-Gln-Ser-Ile-Val-Phe-Gly-Gly-Val-Arg-Ala-Arg-Ser-Gln-Asp-
Leu-Asp-Ala-Ile-Trp-Arg-Gly-Phe-Tyr-Ile-Ala-Gly-Asp-Pro-Ala-Leu-Ala-Tyr-Gly-Tyr-Ala-Gln-
Asp-Gln-Glu-Pro-Asp-Ala-Arg-Gly-Arg-Ile-Arg-Asn-Gly-Ala-Leu-Leu-Arg-Val-Tyr-Val-Pro-Arg-
Ser-Ser-Leu-Pro-Gly-Phe-Tyr-Arg-Thr-Ser-Leu-Thr-Leu-Ala-Ala-Pro-Glu-Ala-Ala-Gly-Glu-Val-
Glu-Arg-Leu-Ile-Gly-His-Pro-Leu-Pro-Leu-Arg-Leu-Asp-Ala-Ile-Thr-Gly-Pro-Glu-Glu-Glu-Gly-
Gly-Arg-Leu-Glu-Thr-Ile-Leu-Gly-Trp-Pro-Leu-Ala-Glu-Arg-Thr-Val-Val-Ile-Pro-Ser-Ala-Ile-Pro-
Thr-Asp-Pro-Arg-Asn-Val-Gly-Gly-Asp-Leu-Asp-Pro-Ser-Ser-Ile-Pro-Asp-Lys-Glu-Gln-Ala-Ile-
Ser-Ala-Leu-Pro-Asp-Tyr-Ala-Ser-Gln-Pro-Gly-Lys-Pro-Pro-Arg-Glu-Glu-Leu
实例五
用PCR方法把融合蛋白PE40部分缺失氨基酸365-380部分，取其氨基酸254-364和氨基酸381-613部分按原顺序首尾相连得到的DNA序列即为蛋白PE38目的基因，然后对目的基因片段LRHR-PE38进行序列测定。验证其基因序列的正确性后，扩增保存，并把目的基因片段，插入表达载体中，再把重组表达载体导入表达宿主菌中，经培养表达，纯化得到目的蛋白LRHR-PE38，用MTT细胞测活法测定LRHR-PE38的杀伤肿瘤细胞的活力。
此序列为LHRH-PE38(序列5)
Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-Gly-Gly-Ser-Leu-Ala-Ala-Leu-Thr-Ala-His-Gln-Ala-
Cys-His-Leu-Pro-Leu-Glu-Thr-Phe-Thr-Arg-His-Arg-Gln-Pro-Arg-Gly-Trp-Glu-Gln-Leu-Glu-Gln
-Cys-Gly-Tyr-Pro-Val-Gln-Arg-Leu-Val-Ala-Leu-Tyr-Leu-Ala-Ala-Arg-Leu-Ser-Trp-Asn-Gln-Val-
Asp-Gln-Val-Ile-Arg-Asn-Ala-Leu-Ala-Ser-Pro-Gly-Ser-Gly-Gly-Asp-Leu-Gly-Glu-Ala-Ile-Arg-
Glu-Gln-Pro-Glu-Gln-Ala-Arg-Leu-Ala-Leu-Thr-Leu-Ala-Ala-Ala-Glu-Ser-Glu-Arg-Phe-Val-Arg-
Gln-Gly-Thr-Gly-Asn-Asp-Glu-Ala-Gly-Ala-Ala-Asn-Gly-Pro-Ala-Asp-Ser-Gly-Asp-Ala-Leu-
Leu-Glu-Arg-Asn-Tyr-Pro-Thr-Gly-Ala-Glu-Phe-Leu-Gly-Asp-Gly-Gly-Asp-Val-Ser-Phe-Ser-Thr-
Arg-Gly-Thr-Gln-Asn-Trp-Thr-Val-Glu-Arg-Leu-Leu-Gln-Ala-His-Arg-Gln-Leu-Glu-Glu-Arg-Gly
-Tyr-Val-Phe-Val-Gly-Tyr-His-Gly-Thr-Phe-Leu-Glu-Ala-Ala-Gln-Ser-Ile-Val-Phe-Gly-Gly-Val-
Arg-Ala-Arg-Ser-Gln-Asp-Leu-Asp-Ala-Ile-Trp-Arg-Gly-Phe-Tyr-Ile-Ala-Gly-Asp-Pro-Ala-Leu-
Ala-Tyr-Gly-Tyr-Ala-Gln-Asp-Gln-Glu-Pro-Asp-Ala-Arg-Gly-Arg-Ile-Arg-Asn-Gly-Ala-Leu-Leu-
Arg-Val-Tyr-Val-Pro-Arg-Ser-Ser-Leu-Pro-Gly-Phe-Tyr-Arg-Thr-Ser-Leu-Thr-Leu-Ala-Ala-Pro-
Glu-Ala-Ala-Gly-Glu-Val-Glu-Arg-Leu-Ile-Gly-His-Pro-Leu-Pro-Leu-Arg-Leu-Asp-Ala-Ile-Thr-
Gly-Pro-Glu-Glu-Glu-Gly-Gly-Arg-Leu-Glu-Thr-Ile-Leu-Gly-Trp-Pro-Leu-Ala-Glu-Arg-Thr-Val-
Val-Ile-Pro-Ser-Ala-Ile-Pro-Thr-Asp-Pro-Arg-Asn-Val-Gly-Gly-Asp-Leu-Asp-Pro-Ser-Ser-Ile-Pro-
Asp-Lys-Glu-Gln-Ala-Ile-Ser-Ala-Leu-Pro-Asp-Tyr-Ala-Ser-Gln-Pro-Gly-Lys-Pro-Pro-Arg-Glu-
Asp-Leu-Lys
实例六
用PCR方法把融合蛋白PE40部分缺失氨基酸254-279，365-380部分，取其氨基酸280-364和氨基酸381-613部分按原顺序首尾位相连得到的DNA序列即为蛋白PE35目的基因，对目的基因片段LHRH-PE35进行序列测定。验证其基因序列的正确性后，扩增保存，并把目的基因片段，插入表达载体中，再把重组表达载体导入表达宿主菌中，经培养表达，纯化得到目的蛋白LHRH-PE35，用MTT细胞测活法测定LRH-PE35的杀伤肿瘤细胞的活力。
此序列为LHRH-PE35(序列6)
Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-Gly-Trp-Glu-Gln-Leu-Glu-Gln-Cys-Gly-Tyr-Pro-Val-
Gln-Arg-Leu-Val-Ala-Leu-Tyr-Leu-Ala-Ala-Arg-Leu-Ser-Trp-Asn-Gln-Val-Asp-Gln-Val-Ile-Arg-
Asn-Ala-Leu-Ala-Ser-Pro-Gly-Ser-Gly-Gly-Asp-Leu-Gly-Glu-Ala-Ile-Arg-Glu-Gln-Pro-Glu-Gln-
Ala-Arg-Leu-Ala-Leu-Thr-Leu-Ala-Ala-Ala-Glu-Ser-Glu-Arg-Phe-Val-Arg-Gln-Gly-Thr-Gly-Asn
-Asp-Glu-Ala-Gly-Ala-Ala-Asn-Gly-Pro-Ala-Asp-Ser-Gly-Asp-Ala-Leu-Leu-Glu-Arg-Asn-Tyr-
Pro-Thr-Gly-Ala-Glu-Phe-Leu-Gly-Asp-Gly-Gly-Asp-Val-Ser-Phe-Ser-Thr-Arg-Gly-Thr-Gln-Asn-
Trp-Thr-Val-Glu-Arg-Leu-Leu-Gln-Ala-His-Arg-Gln-Leu-Glu-Glu-Arg-Gly-Tyr-Val-Phe-Val-Gly-
Tyr-His-Gly-Thr-Phe-Leu-Glu-Ala-Ala-Gln-Ser-Ile-Val-Phe-Gly-Gly-Val-Arg-Ala-Arg-Ser-Gln-
Asp-Leu-Asp-Ala-Ile-Trp-Arg-Gly-Phe-Tyr-Ile-Ala-Gly-Asp-Pro-Ala-Leu-Ala-Tyr-Gly-Tyr-Ala-
Gln-Asp-Gln-Glu-Pro-Asp-Ala-Arg-Gly-Arg-Ile-Arg-Asn-Gly-Ala-Leu-Leu-Arg-Val-Tyr-Val-Pro-
Arg-Ser-Ser-Leu-Pro-Gly-Phe-Tyr-Arg-Thr-Ser-Leu-Thr-Leu-Ala-Ala-Pro-Glu-Ala-Ala-Gly-Glu-
Val-Glu-Arg-Leu-Ile-Gly-His-Pro-Leu-Pro-Leu-Arg-Leu-Asp-Ala-Ile-Thr-Gly-Pro-Glu-Glu-Glu-
Gly-Gly-Arg-Leu-Glu-Thr-Ile-Leu-Gly-Trp-Pro-Leu-Ala-Glu-Arg-Thr-Val-Val-Ile-Pro-Ser-Ala-Ile
-Pro-Thr-Asp-Pro-Arg-Asn-Val-Gly-Gly-Asp-Leu-Asp-Pro-Ser-Ser-Ile-Pro-Asp-Lys-Glu-Gln-Ala-
Ile-Ser-Ala-Leu-Pro-Asp-Tyr-Ala-Ser-Gln-Pro-Gly-Lys-Pro-Pro-Arg-Glu-Asp-Leu-Lys
实例七
从pLHRH-PE35载体用PCR方法把融合蛋白PE35部分C末端4个氨基酸突变为KDEL，得到目的基因片段LHRH-PE35(KDEL)进行序列测定。验证其基因序列的正确性后，扩增保存，从该载体上切取下目的基因片段，插入表达载体中，再把重组表达载体导入表达宿主菌中，经培养表达，纯化得到目的蛋白LHRH-PE35(KDEL)，用MTT细胞测活法测定LHRH-PE35(KDEL)的杀伤肿瘤细胞的活力。
此序列为LHRH-PE35(KDEL)(序列7)
Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-Gly-Trp-Glu-Gln-Leu-Glu-Gln-Cys-Gly-Tyr-Pro-Val-
Gln-Arg-Leu-Val-Ala-Leu-Tyr-Leu-Ala-Ala-Arg-Leu-Ser-Trp-Asn-Gln-Val-Asp-Gln-Val-Ile-Arg-
Asn-Ala-Leu-Ala-Ser-Pro-Gly-Ser-Gly-Gly-Asp-Leu-Gly-Glu-Ala-Ile-Arg-Glu-Gln-Pro-Glu-Gln-
Ala-Arg-Leu-Ala-Leu-Thr-Leu-Ala-Ala-Ala-Glu-Ser-Glu-Arg-Phe-Val-Arg-Gln-Gly-Thr-Gly-Asn
-Asp-Glu-Ala-Gly-Ala-Ala-Asn-Gly-Pro-Ala-Asp-Ser-Gly-Asp-Ala-Leu-Leu-Glu-Arg-Asn-Tyr-
Pro-Thr-Gly-Ala-Glu-Phe-Leu-Gly-Asp-Gly-Gly-Asp-Val-Ser-Phe-Ser-Thr-Arg-Gly-Thr-Gln-Asn-
Trp-Thr-Val-Glu-Arg-Leu-Leu-Gln-Ala-His-Arg-Gln-Leu-Glu-Glu-Arg-Gly-Tyr-Val-Phe-Val-Gly-
Tyr-His-Gly-Thr-Phe-Leu-Glu-Ala-Ala-Gln-Ser-Ile-Val-Phe-Gly-Gly-Val-Arg-Ala-Arg-Ser-Gln-
Asp-Leu-Asp-Ala-Ile-Trp-Arg-Gly-Phe-Tyr-Ile-Ala-Gly-Asp-Pro-Ala-Leu-Ala-Tyr-Gly-Tyr-Ala-
Gln-Asp-Gln-Glu-Pro-Asp-Ala-Arg-Gly-Arg-Ile-Arg-Asn-Gly-Ala-Leu-Leu-Arg-Val-Tyr-Val-Pro-
Arg-Ser-Ser-Leu-Pro-Gly-Phe-Tyr-Arg-Thr-Ser-Leu-Thr-Leu-Ala-Ala-Pro-Glu-Ala-Ala-Gly-Glu-
Val-Glu-Arg-Leu-Ile-Gly-His-Pro-Leu-Pro-Leu-Arg-Leu-Asp-Ala-Ile-Thr-Gly-Pro-Glu-Glu-Glu-
Gly-Gly-Arg-Leu-Glu-Thr-Ile-Leu-Gly-Trp-Pro-Leu-Ala-Glu-Arg-Thr-Val-Val-Ile-Pro-Ser-Ala-Ile
-Pro-Thr-Asp-Pro-Arg-Asn-Val-Gly-Gly-Asp-Leu-Asp-Pro-Ser-Ser-Ile-Pro-Asp-Lys-Glu-Gln-Ala-
Ile-Ser-Ala-Leu-Pro-Asp-Tyr-Ala-Ser-Gln-Pro-Gly-Lys-Pro-Pro-Lys-Asp-Glu-Leu
实例八
从pLHRH-PE40载体上，用PCR方法把融合蛋白LHRH部分N末端第6位氨基酸突变为Trp，得到目的基因片段LHRH(Trp6)-PE40进行序列测定。验证其基因序列的正确性后，扩增保存，从该载体上切取下目的基因片段，插入表达载体中，再把重组表达载体导入表达宿主菌中，经培养表达，纯化得到目的蛋白LHRH(Trp6)-PE40，用MTT细胞测活法测定LHRH(Trp6)-PE40的杀伤肿瘤细胞的活力。
此序列为LHRH(Trp6)-PE40(序列8)
Glu-His-Trp-Ser-Tyr-Trp-Leu-Arg-Pro-Gly-Gly-Gly-Ser-Leu-Ala-Ala-Leu-Thr-Ala-His-Gln-Ala-
Cys-His-Leu-Pro-Leu-Glu-Thr-Phe-Thr-Arg-His-Arg-Gln-Pro-Arg-Gly-Trp-Glu-Gln-Leu-Glu-Gln
-Cys-Gly-Tyr-Pro-Val-Gln-Arg-Leu-Val-Ala-Leu-Tyr-Leu-Ala-Ala-Arg-Leu-Ser-Trp-Asn-Gln-Val-
Asp-Gln-Val-Ile-Arg-Asn-Ala-Leu-Ala-Ser-Pro-Gly-Ser-Gly-Gly-Asp-Leu-Gly-Glu-Ala-Ile-Arg-
Glu-Gln-Pro-Glu-Gln-Ala-Arg-Leu-Ala-Leu-Thr-Leu-Ala-Ala-Ala-Glu-Ser-Glu-Arg-Phe-Val-Arg-
Gln-Gly-Thr-Gly-Asn-Asp-Glu-Ala-Gly-Ala-Ala-Asn-Ala-Asp-Val-Val-Ser-Leu-Thr-Cys-Pro-Val-
Ala-Ala-Gly-Glu-Cys-Ala-Gly-Pro-Ala-Asp-Ser-Gly-Asp-Ala-Leu-Leu-Glu-Arg-Asn-Tyr-Pro-Thr
-Gly-Ala-Glu-Phe-Leu-Gly-Asp-Gly-Gly-Asp-Val-Ser-Phe-Ser-Thr-Arg-Gly-Thr-Gln-Asn-Trp-
Thr-Val-Glu-Arg-Leu-Leu-Gln-Ala-His-Arg-Gln-Leu-Glu-Glu-Arg-Gly-Tyr-Val-Phe-Val-Gly-Tyr-
His-Gly-Thr-Phe-Leu-Glu-Ala-Ala-Gln-Ser-Ile-Val-Phe-Gly-Gly-Val-Arg-Ala-Arg-Ser-Gln-Asp-
Leu-Asp-Ala-Ile-Trp-Arg-Gly-Phe-Tyr-Ile-Ala-Gly-Asp-Pro-Ala-Leu-Ala-Tyr-Gly-Tyr-Ala-Gln-
Asp-Gln-Glu-Pro-Asp-Ala-Arg-Gly-Arg-Ile-Arg-Asn-Gly-Ala-Leu-Leu-Arg-Val-Tyr-Val-Pro-Arg-
Ser-Ser-Leu-Pro-Gly-Phe-Tyr-Arg-Thr-Ser-Leu-Thr-Leu-Ala-Ala-Pro-Glu-Ala-Ala-Gly-Glu-Val-
Glu-Arg-Leu-Ile-Gly-His-Pro-Leu-Pro-Leu-Arg-Leu-Asp-Ala-Ile-Thr-Gly-Pro-Glu-Glu-Glu-Gly-
Gly-Arg-Leu-Glu-Thr-Ile-Leu-Gly-Trp-Pro-Leu-Ala-Glu-Arg-Thr-Val-Val-Ile-Pro-Ser-Ala-Ile-Pro-
Thr-Asp-Pro-Arg-Asn-Val-Gly-Gly-Asp-Leu-Asp-Pro-Ser-Ser-Ile-Pro-Asp-Lys-Glu-Gln-Ala-Ile-
Ser-Ala-Leu-Pro-Asp-Tyr-Ala-Ser-Gln-Pro-Gly-Lys-Pro-Pro-Arg-Glu-Asp-Leu-Lys
实例九
从LHRH(Trp6)-PE40载体用PCR方法把融合蛋白PE40部分C末端5个氨基酸突变为KDEL，得到目的基因片段LHRH(Trp6)-PE40(KDEL)进行序列测定。验证其基因序列的正确性后，扩增保存，从该载体上切取下目的基因片段，插入表达载体中，再把重组表达载体导入表达宿主菌中，经培养表达，纯化得到目的蛋白LHRH(Trp6)-PE40(KDEL)，用MTT细胞测活法测定LHRH(Trp6)-PE40(KDEL)的杀伤肿瘤细胞的活力。
此序列为LHRH(Trp6)-PE40(KDEL)(序列9)
Glu-His-Trp-Ser-Tyr-Trp-Leu-Arg-Pro-Gly-Gly-Gly-Ser-Leu-Ala-Ala-Leu-Thr-Ala-His-Gln-Ala-
Cys-His-Leu-Pro-Leu-Glu-Thr-Phe-Thr-Arg-His-Arg-Gln-Pro-Arg-Gly-Trp-Glu-Gln-Leu-Glu-Gln
-Cys-Gly-Tyr-Pro-Val-Gln-Arg-Leu-Val-Ala-Leu-Tyr-Leu-Ala-Ala-Arg-Leu-Ser-Trp-Asn-Gln-Val-
Asp-Gln-Val-Ile-Arg-Asn-Ala-Leu-Ala-Ser-Pro-Gly-Ser-Gly-Gly-Asp-Leu-Gly-Glu-Ala-Ile-Arg-
Glu-Gln-Pro-Glu-Gln-Ala-Arg-Leu-Ala-Leu-Thr-Leu-Ala-Ala-Ala-Glu-Ser-Glu-Arg-Phe-Val-Arg-
Gln-Gly-Thr-Gly-Asn-Asp-Glu-Ala-Gly-Ala-Ala-Asn-Ala-Asp-Val-Val-Ser-Leu-Thr-Cys-Pro-Val-
Ala-Ala-Gly-Glu-Cys-Ala-Gly-Pro-Ala-Asp-Ser-Gly-Asp-Ala-Leu-Leu-Glu-Arg-Asn-Tyr-Pro-Thr
-Gly-Ala-Glu-Phe-Leu-Gly-Asp-Gly-Gly-Asp-Val-Ser-Phe-Ser-Thr-Arg-Gly-Thr-Gln-Asn-Trp-
Thr-Val-Glu-Arg-Leu-Leu-Gln-Ala-His-Arg-Gln-Leu-Glu-Glu-Arg-Gly-Tyr-Val-Phe-Val-Gly-Tyr-
His-Gly-Thr-Phe-Leu-Glu-Ala-Ala-Gln-Ser-Ile-Val-Phe-Gly-Gly-Val-Arg-Ala-Arg-Ser-Gln-Asp-
Leu-Asp-Ala-Ile-Trp-Arg-Gly-Phe-Tyr-Ile-Ala-Gly-Asp-Pro-Ala-Leu-Ala-Tyr-Gly-Tyr-Ala-Gln-
Asp-Gln-Glu-Pro-Asp-Ala-Arg-Gly-Arg-Ile-Arg-Asn-Gly-Ala-Leu-Leu-Arg-Val-Tyr-Val-Pro-Arg-
Ser-Ser-Leu-Pro-Gly-Phe-Tyr-Arg-Thr-Ser-Leu-Thr-Leu-Ala-Ala-Pro-Glu-Ala-Ala-Gly-Glu-Val-
Glu-Arg-Leu-Ile-Gly-His-Pro-Leu-Pro-Leu-Arg-Leu-Asp-Ala-Ile-Thr-Gly-Pro-Glu-Glu-Glu-Gly-
Gly-Arg-Leu-Glu-Thr-Ile-Leu-Gly-Trp-Pro-Leu-Ala-Glu-Arg-Thr-Val-Val-Ile-Pro-Ser-Ala-Ile-Pro-
Thr-Asp-Pro-Arg-Asn-Val-Gly-Gly-Asp-Leu-Asp-Pro-Ser-Ser-Ile-Pro-Asp-Lys-Glu-Gln-Ala-Ile-
Ser-Ala-Leu-Pro-Asp-Tyr-Ala-Ser-Gln-Pro-Gly-Lys-Pro-Pro-Lys-Asp-Glu-Leu
实例十
从pLHRH-PE38载体用PCR方法把融合蛋白LHRH部分N末端第6位氨基酸突变为Trp，得到目的基因片段LHRH(Trp6)-PE38进行序列测定。验证其基因序列的正确性后，扩增保存，从该载体上切取下目的基因片段，插入表达载体中，再把重组表达载体导入表达宿主菌中，经培养表达，纯化得到目的蛋白LHRH(Trp6)-PE38，用MTT细胞测活法测定LHRH(Trp6)-PE38的杀伤肿瘤细胞的活力。
此序列为LHRH(Trp6)-PE38(序列10)
Glu-His-Trp-Ser-Tyr-Trp-Leu-Arg-Pro-Gly-Gly-Gly-Ser-Leu-Ala-Ala-Leu-Thr-Ala-His-Gln-Ala-
Cys-His-Leu-Pro-Leu-Glu-Thr-Phe-Thr-Arg-His-Arg-Gln-Pro-Arg-Gly-Trp-Glu-Gln-Leu-Glu-Gln
-Cys-Gly-Tyr-Pro-Val-Gln-Arg-Leu-Val-Ala-Leu-Tyr-Leu-Ala-Ala-Arg-Leu-Ser-Trp-Asn-Gln-Val-
Asp-Gln-Val-Ile-Arg-Asn-Ala-Leu-Ala-Ser-Pro-Gly-Ser-Gly-Gly-Asp-Leu-Gly-Glu-Ala-Ile-Arg-
Glu-Gln-Pro-Glu-Gln-Ala-Arg-Leu-Ala-Leu-Thr-Leu-Ala-Ala-Ala-Glu-Ser-Glu-Arg-Phe-Val-Arg-
Gln-Gly-Thr-Gly-Asn-Asp-Glu-Ala-Gly-Ala-Ala-Asn-Gly-Pro-Ala-Asp-Ser-Gly-Asp-Ala-Leu-
Leu-Glu-Arg-Asn-Tyr-Pro-Thr-Gly-Ala-Glu-Phe-Leu-Gly-Asp-Gly-Gly-Asp-Val-Ser-Phe-Ser-Thr-
Arg-Gly-Thr-Gln-Asn-Trp-Thr-Val-Glu-Arg-Leu-Leu-Gln-Ala-His-Arg-Gln-Leu-Glu-Glu-Arg-Gly
-Tyr-Val-Phe-Val-Gly-Tyr-His-Gly-Thr-Phe-Leu-Glu-Ala-Ala-Gln-Ser-Ile-Val-Phe-Gly-Gly-Val-
Arg-Ala-Arg-Ser-Gln-Asp-Leu-Asp-Ala-Ile-Trp-Arg-Gly-Phe-Tyr-Ile-Ala-Gly-Asp-Pro-Ala-Leu-
Ala-Tyr-Gly-Tyr-Ala-Gln-Asp-Gln-Glu-Pro-Asp-Ala-Arg-Gly-Arg-Ile-Arg-Asn-Gly-Ala-Leu-Leu-
Arg-Val-Tyr-Val-Pro-Arg-Ser-Ser-Leu-Pro-Gly-Phe-Tyr-Arg-Thr-Ser-Leu-Thr-Leu-Ala-Ala-Pro-
Glu-Ala-Ala-Gly-Glu-Val-Glu-Arg-Leu-Ile-Gly-His-Pro-Leu-Pro-Leu-Arg-Leu-Asp-Ala-Ile-Thr-
Gly-Pro-Glu-Glu-Glu-Gly-Gly-Arg-Leu-Glu-Thr-Ile-Leu-Gly-Trp-Pro-Leu-Ala-Glu-Arg-Thr-Val-
Val-Ile-Pro-Ser-Ala-Ile-Pro-Thr-Asp-Pro-Arg-Asn-Val-Gly-Gly-Asp-Leu-Asp-Pro-Ser-Ser-Ile-Pro-
Asp-Lys-Glu-Gln-Ala-Ile-Ser-Ala-Leu-Pro-Asp-Tyr-Ala-Ser-Gln-Pro-Gly-Lys-Pro-Pro-Arg-Glu-
Asp-Leu-Lys
实例十一
从LHRH(Trp6)-PE38载体用PCR方法把融合蛋白PE38部分C末端5个氨基酸突变为KDEL，得到目的基因片段LHRH(Trp6)-PE38(KDEL)进行序列测定。验证其基因序列的正确性后，扩增保存，从该载体上切取下目的基因片段，插入表达载体中，再把重组表达载体导入表达宿主菌中，经培养表达，纯化得到目的蛋白LHRH(Trp6)-PE38(KDEL)，用MTT细胞测活法测定LHRH(Trp6)-PE38(KDEL)的杀伤肿瘤细胞的活力。
此序列为LHRH(Trp6)-PE38(KDEL)(序列11)
Glu-His-Trp-Ser-Tyr-Trp-Leu-Arg-Pro-Gly-Gly-Gly-Ser-Leu-Ala-Ala-Leu-Thr-Ala-His-Gln-Ala-
Cys-His-Leu-Pro-Leu-Glu-Thr-Phe-Thr-Arg-His-Arg-Gln-Pro-Arg-Gly-Trp-Glu-Gln-Leu-Glu-Gln
-Cys-Gly-Tyr-Pro-Val-Gln-Arg-Leu-Val-Ala-Leu-Tyr-Leu-Ala-Ala-Arg-Leu-Ser-Trp-Asn-Gln-Val-
Asp-Gln-Val-Ile-Arg-Asn-Ala-Leu-Ala-Ser-Pro-Gly-Ser-Gly-Gly-Asp-Leu-Gly-Glu-Ala-Ile-Arg-
Glu-Gln-Pro-Glu-Gln-Ala-Arg-Leu-Ala-Leu-Thr-Leu-Ala-Ala-Ala-Glu-Ser-Glu-Arg-Phe-Val-Arg-
Gln-Gly-Thr-Gly-Asn-Asp-Glu-Ala-Gly-Ala-Ala-Asn-Gly-Pro-Ala-Asp-Ser-Gly-Asp-Ala-Leu-
Leu-Glu-Arg-Asn-Tyr-Pro-Thr-Gly-Ala-Glu-Phe-Leu-Gly-Asp-Gly-Gly-Asp-Val-Ser-Phe-Ser-Thr-
Arg-Gly-Thr-Gln-Asn-Trp-Thr-Val-Glu-Arg-Leu-Leu-Gln-Ala-His-Arg-Gln-Leu-Glu-Glu-Arg-Gly
-Tyr-Val-Phe-Val-Gly-Tyr-His-Gly-Thr-Phe-Leu-Glu-Ala-Ala-Gln-Ser-Ile-Val-Phe-Gly-Gly-Val-
Arg-Ala-Arg-Ser-Gln-Asp-Leu-Asp-Ala-Ile-Trp-Arg-Gly-Phe-Tyr-Ile-Ala-Gly-Asp-Pro-Ala-Leu-
Ala-Tyr-Gly-Tyr-Ala-Gln-Asp-Gln-Glu-Pro-Asp-Ala-Arg-Gly-Arg-Ile-Arg-Asn-Gly-Ala-Leu-Leu-
Arg-Val-Tyr-Val-Pro-Arg-Ser-Ser-Leu-Pro-Gly-Phe-Tyr-Arg-Thr-Ser-Leu-Thr-Leu-Ala-Ala-Pro-
Glu-Ala-Ala-Gly-Glu-Val-Glu-Arg-Leu-Ile-Gly-His-Pro-Leu-Pro-Leu-Arg-Leu-Asp-Ala-Ile-Thr-
Gly-Pro-Glu-Glu-Glu-Gly-Gly-Arg-Leu-Glu-Thr-Ile-Leu-Gly-Trp-Pro-Leu-Ala-Glu-Arg-Thr-Val-
Val-Ile-Pro-Ser-Ala-Ile-Pro-Thr-Asp-Pro-Arg-Asn-Val-Gly-Gly-Asp-Leu-Asp-Pro-Ser-Ser-Ile-Pro-
Asp-Lys-Glu-Gln-Ala-Ile-Ser-Ala-Leu-Pro-Asp-Tyr-Ala-Ser-Gln-Pro-Gly-Lys-Pro-Pro-Lys-Asp-
Glu-Leu
实例十二
从pLHRH-PE35载体用PCR方法把融合蛋白LHRH部分N末端第6位氨基酸突变为Ala，得到目的基因片段LHRH(Ala6)-PE35进行序列测定。验证其基因序列的正确性后，扩增保存，从该载体上切取下目的基因片段，插入表达载体中，再把重组表达载体导入表达宿主菌中，经培养表达，纯化得到目的蛋白LHRH(Ala6)-PE35，用MTT细胞测活法测定LHRH(Ala6)-PE35的杀伤肿瘤细胞的活力。
此序列为LHRH(Ala6)-PE35(序列12)
Glu-His-Trp-Ser-Tyr-Ala-Leu-Arg-Pro-Gly-Gly-Trp-Glu-Gln-Leu-Glu-Gln-Cys-Gly-Tyr-Pro-Val-
Gln-Arg-Leu-Val-Ala-Leu-Tyr-Leu-Ala-Ala-Arg-Leu-Ser-Trp-Asn-Gln-Val-Asp-Gln-Val-Ile-Arg-
Asn-Ala-Leu-Ala-Ser-Pro-Gly-Ser-Gly-Gly-Asp-Leu-Gly-Glu-Ala-Ile-Arg-Glu-Gln-Pro-Glu-Gln-
Ala-Arg-Leu-Ala-Leu-Thr-Leu-Ala-Ala-Ala-Glu-Ser-Glu-Arg-Phe-Val-Arg-Gln-Gly-Thr-Gly-Asn
-Asp-Glu-Ala-Gly-Ala-Ala-Asn-Gly-Pro-Ala-Asp-Ser-Gly-Asp-Ala-Leu-Leu-Glu-Arg-Asn-Tyr-
Pro-Thr-Gly-Ala-Glu-Phe-Leu-Gly-Asp-Gly-Gly-Asp-Val-Ser-Phe-Ser-Thr-Arg-Gly-Thr-Gln-Asn-
Trp-Thr-Val-Glu-Arg-Leu-Leu-Gln-Ala-His-Arg-Gln-Leu-Glu-Glu-Arg-Gly-Tyr-Val-Phe-Val-Gly-
Tyr-His-Gly-Thr-Phe-Leu-Glu-Ala-Ala-Gln-Ser-Ile-Val-Phe-Gly-Gly-Val-Arg-Ala-Arg-Ser-Gln-
Asp-Leu-Asp-Ala-Ile-Trp-Arg-Gly-Phe-Tyr-Ile-Ala-Gly-Asp-Pro-Ala-Leu-Ala-Tyr-Gly-Tyr-Ala-
Gln-Asp-Gln-Glu-Pro-Asp-Ala-Arg-Gly-Arg-Ile-Arg-Asn-Gly-Ala-Leu-Leu-Arg-Val-Tyr-Val-Pro-
Arg-Ser-Ser-Leu-Pro-Gly-Phe-Tyr-Arg-Thr-Ser-Leu-Thr-Leu-Ala-Ala-Pro-Glu-Ala-Ala-Gly-Glu-
Val-Glu-Arg-Leu-Ile-Gly-His-Pro-Leu-Pro-Leu-Arg-Leu-Asp-Ala-Ile-Thr-Gly-Pro-Glu-Glu-Glu-
Gly-Gly-Arg-Leu-Glu-Thr-Ile-Leu-Gly-Trp-Pro-Leu-Ala-Glu-Arg-Thr-Val-Val-Ile-Pro-Ser-Ala-Ile
-Pro-Thr-Asp-Pro-Arg-Asn-Val-Gly-Gly-Asp-Leu-Asp-Pro-Ser-Ser-Ile-Pro-Asp-Lys-Glu-Gln-Ala-
Ile-Ser-Ala-Leu-Pro-Asp-Tyr-Ala-Ser-Gln-Pro-Gly-Lys-Pro-Pro-Arg-Glu-Asp-Leu-Lys
实例十三
从LHRH(Ala6)-PE35载体用PCR方法把融合蛋白PE35部分C末端5个氨基酸突变为KDEL，得到目的基因片段LHRH(Ala6)-PE35(KDEL)进行序列测定。验证其基因序列的正确性后，扩增保存，从该载体上切取下目的基因片段，插入表达载体中，再把重组表达载体导入表达宿主菌中，经培养表达，纯化得到目的蛋白LHRH(Ala6)-PE35(KDEL)，用MTT细胞测活法测定LHRH(Ala6)-PE35(KDEL)的杀伤肿瘤细胞的活力。
此序列为LHRH(Ala6)-PE35(KDEL)(序列13)
Glu-His-Trp-Ser-Tyr-Ala-Leu-Arg-Pro-Gly-Gly-Trp-Glu-Gln-Leu-Glu-Gln-Cys-Gly-Tyr-Pro-Val-
Gln-Arg-Leu-Val-Ala-Leu-Tyr-Leu-Ala-Ala-Arg-Leu-Ser-Trp-Asn-Gln-Val-Asp-Gln-Val-Ile-Arg-
Asn-Ala-Leu-Ala-Ser-Pro-Gly-Ser-Gly-Gly-Asp-Leu-Gly-Glu-Ala-Ile-Arg-Glu-Gln-Pro-Glu-Gln-
Ala-Arg-Leu-Ala-Leu-Thr-Leu-Ala-Ala-Ala-Glu-Ser-Glu-Arg-Phe-Val-Arg-Gln-Gly-Thr-Gly-Asn
-Asp-Glu-Ala-Gly-Ala-Ala-Asn-Gly-Pro-Ala-Asp-Ser-Gly-Asp-Ala-Leu-Leu-Glu-Arg-Asn-Tyr-
Pro-Thr-Gly-Ala-Glu-Phe-Leu-Gly-Asp-Gly-Gly-Asp-Val-Ser-Phe-Ser-Thr-Arg-Gly-Thr-Gln-Asn-
Trp-Thr-Val-Glu-Arg-Leu-Leu-Gln-Ala-His-Arg-Gln-Leu-Glu-Glu-Arg-Gly-Tyr-Val-Phe-Val-Gly-
Tyr-His-Gly-Thr-Phe-Leu-Glu-Ala-Ala-Gln-Ser-Ile-Val-Phe-Gly-Gly-Val-Arg-Ala-Arg-Ser-Gln-
Asp-Leu-Asp-Ala-Ile-Trp-Arg-Gly-Phe-Tyr-Ile-Ala-Gly-Asp-Pro-Ala-Leu-Ala-Tyr-Gly-Tyr-Ala-
Gln-Asp-Gln-Glu-Pro-Asp-Ala-Arg-Gly-Arg-Ile-Arg-Asn-Gly-Ala-Leu-Leu-Arg-Val-Tyr-Val-Pro-
Arg-Ser-Ser-Leu-Pro-Gly-Phe-Tyr-Arg-Thr-Ser-Leu-Thr-Leu-Ala-Ala-Pro-Glu-Ala-Ala-Gly-Glu-
Val-Glu-Arg-Leu-Ile-Gly-His-Pro-Leu-Pro-Leu-Arg-Leu-Asp-Ala-Ile-Thr-Gly-Pro-Glu-Glu-Glu-
Gly-Gly-Arg-Leu-Glu-Thr-Ile-Leu-Gly-Trp-Pro-Leu-Ala-Glu-Arg-Thr-Val-Val-Ile-Pro-Ser-Ala-Ile
-Pro-Thr-Asp-Pro-Arg-Asn-Val-Gly-Gly-Asp-Leu-Asp-Pro-Ser-Ser-Ile-Pro-Asp-Lys-Glu-Gln-Ala-
Ile-Ser-Ala-Leu-Pro-Asp-Tyr-Ala-Ser-Gln-Pro-Gly-Lys-Pro-Pro-Lys-Asp-Glu-Leu
实例十四
从LHRH(Ala6)-PE35载体用PCR方法把融合蛋白PE35部分C末端5个氨基酸突变为RQEL，得到目的基因片段LHRH(Ala6)-PE35(RQEL)进行序列测定。验证其基因序列的正确性后，扩增保存，从该载体上切取下目的基因片段，插入表达载体中，再把重组表达载体导入表达宿主菌中，经培养表达，纯化得到目的蛋白LHRH(Ala6)-PE35(RQEL)，用MTT细胞测活法测定LHRH(Ala6)-PE35(RQEL)的杀伤肿瘤细胞的活力。
此序列为LHRH(Ala6)-PE35(RQEL)(序列14)
Glu-His-Trp-Ser-Tyr-Ala-Leu-Arg-Pro-Gly-Gly-Trp-Glu-Gln-Leu-Glu-Gln-Cys-Gly-Tyr-Pro-Val-
Gln-Arg-Leu-Val-Ala-Leu-Tyr-Leu-Ala-Ala-Arg-Leu-Ser-Trp-Asn-Gln-Val-Asp-Gln-Val-Ile-Arg-
Asn-Ala-Leu-Ala-Ser-Pro-Gly-Ser-Gly-Gly-Asp-Leu-Gly-Glu-Ala-Ile-Arg-Glu-Gln-Pro-Glu-Gln-
Ala-Arg-Leu-Ala-Leu-Thr-Leu-Ala-Ala-Ala-Glu-Ser-Glu-Arg-Phe-Val-Arg-Gln-Gly-Thr-Gly-Asn
-Asp-Glu-Ala-Gly-Ala-Ala-Asn-Gly-Pro-Ala-Asp-Ser-Gly-Asp-Ala-Leu-Leu-Glu-Arg-Asn-Tyr-
Pro-Thr-Gly-Ala-Glu-Phe-Leu-Gly-Asp-Gly-Gly-Asp-Val-Ser-Phe-Ser-Thr-Arg-Gly-Thr-Gln-Asn-
Trp-Thr-Val-Glu-Arg-Leu-Leu-Gln-Ala-His-Arg-Gln-Leu-Glu-Glu-Arg-Gly-Tyr-Val-Phe-Val-Gly-
Tyr-His-Gly-Thr-Phe-Leu-Glu-Ala-Ala-Gln-Ser-Ile-Val-Phe-Gly-Gly-Val-Arg-Ala-Arg-Ser-Gln-
Asp-Leu-Asp-Ala-Ile-Trp-Arg-Gly-Phe-Tyr-Ile-Ala-Gly-Asp-Pro-Ala-Leu-Ala-Tyr-Gly-Tyr-Ala-
Gln-Asp-Gln-Glu-Pro-Asp-Ala-Arg-Gly-Arg-Ile-Arg-Asn-Gly-Ala-Leu-Leu-Arg-Val-Tyr-Val-Pro-
Arg-Ser-Ser-Leu-Pro-Gly-Phe-Tyr-Arg-Thr-Ser-Leu-Thr-Leu-Ala-Ala-Pro-Glu-Ala-Ala-Gly-Glu-
Val-Glu-Arg-Leu-Ile-Gly-His-Pro-Leu-Pro-Leu-Arg-Leu-Asp-Ala-Ile-Thr-Gly-Pro-Glu-Glu-Glu-
Gly-Gly-Arg-Leu-Glu-Thr-Ile-Leu-Gly-Trp-Pro-Leu-Ala-Glu-Arg-Thr-Val-Val-Ile-Pro-Ser-Ala-Ile
-Pro-Thr-Asp-Pro-Arg-Asn-Val-Gly-Gly-Asp-Leu-Asp-Pro-Ser-Ser-Ile-Pro-Asp-Lys-Glu-Gln-Ala-
Ile-Ser-Ala-Leu-Pro-Asp-Tyr-Ala-Ser-Gln-Pro-Gly-Lys-Pro-Pro-Arg-Gln-Glu-Leu
实例十五
从cDNA文库中用PCR方法得到LHRH与PE40的基因片段后，用PCR与基因重组方法把PE40的C末端和LHRH的N末端相连，并在LHRH的C末端加上KDEL，获得重组基因载体pPE40-LHRH-KDEL，对插入载体的基因片段PE40-LHRH-KDEL进行序列测定。验证其基因序列的正确性后，扩增保存，从该载体上切取下目的基因片段，插入表达载体中，再把重组表达载体导入表达宿主菌中，经培养表达，纯化得到目的蛋白PE40-LHRH-KDEL，用MTT细胞测活法测定PE40-LHRH-KDEL的杀伤肿瘤细胞的活力。
此序列为PE40-LHRH-KDEL(序列15)
Gly-Gly-Ser-Leu-Ala-Ala-Leu-Thr-Ala-His-Gln-Ala-Cys-His-Leu-Pro-Leu-Glu-Thr-Phe-Thr-Arg-
His-Arg-Gln-Pro-Arg-Gly-Trp-Glu-Gln-Leu-Glu-Gln-Cys-Gly-Tyr-Pro-Val-Gln-Arg-Leu-Val-Ala-
Leu-Tyr-Leu-Ala-Ala-Arg-Leu-Ser-Trp-Asn-Gln-Val-Asp-Gln-Val-Ile-Arg-Asn-Ala-Leu-Ala-Ser-
Pro-Gly-Ser-Gly-Gly-Asp-Leu-Gly-Glu-Ala-Ile-Arg-Glu-Gln-Pro-Glu-Gln-Ala-Arg-Leu-Ala-Leu-
Thr-Leu-Ala-Ala-Ala-Glu-Ser-Glu-Arg-Phe-Val-Arg-Gln-Gly-Thr-Gly-Asn-Asp-Glu-Ala-Gly-Ala
-Ala-Asn-Ala-Asp-Val-Val-Ser-Leu-Thr-Cys-Pro-Val-Ala-Ala-Gly-Glu-Cys-Ala-Gly-Pro-Ala-Asp-
Ser-Gly-Asp-Ala-Leu-Leu-Glu-Arg-Asn-Tyr-Pro-Thr-Gly-Ala-Glu-Phe-Leu-Gly-Asp-Gly-Gly-
Asp-Val-Ser-Phe-Ser-Thr-Arg-Gly-Thr-Gln-Asn-Trp-Thr-Val-Glu-Arg-Leu-Leu-Gln-Ala-His-Arg-
Gln-Leu-Glu-Glu-Arg-Gly-Tyr-Val-Phe-Val-Gly-Tyr-His-Gly-Thr-Phe-Leu-Glu-Ala-Ala-Gln-Ser-
Ile-Val-Phe-Gly-Gly-Val-Arg-Ala-Arg-Ser-Gln-Asp-Leu-Asp-Ala-Ile-Trp-Arg-Gly-Phe-Tyr-Ile-
Ala-Gly-Asp-Pro-Ala-Leu-Ala-Tyr-Gly-Tyr-Ala-Gln-Asp-Gln-Glu-Pro-Asp-Ala-Arg-Gly-Arg-Ile-
Arg-Asn-Gly-Ala-Leu-Leu-Arg-Val-Tyr-Val-Pro-Arg-Ser-Ser-Leu-Pro-Gly-Phe-Tyr-Arg-Thr-Ser-
Leu-Thr-Leu-Ala-Ala-Pro-Glu-Ala-Ala-Gly-Glu-Val-Glu-Arg-Leu-Ile-Gly-His-Pro-Leu-Pro-Leu-
Arg-Leu-Asp-Ala-Ile-Thr-Gly-Pro-Glu-Glu-Glu-Gly-Gly-Arg-Leu-Glu-Thr-Ile-Leu-Gly-Trp-Pro-
Leu-Ala-Glu-Arg-Thr-Val-Val-Ile-Pro-Ser-Ala-Ile-Pro-Thr-Asp-Pro-Arg-Asn-Val-Gly-Gly-Asp-
Leu-Asp-Pro-Ser-Ser-Ile-Pro-Asp-Lys-Glu-Gln-Ala-Ile-Ser-Ala-Leu-Pro-Asp-Tyr-Ala-Ser-Gln-Pro
-Gly-Lys-Pro-Pro-Arg-Glu-Asp-Leu-Lys-Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-Lys-Asp-
Glu-Leu
实例十六
从pPE40-LHRH-KDEL载体上，用PCR方法把融合蛋白PE40部分缺失氨基酸365-380部分，取其氨基酸254-364和氨基酸381-613部分及LHRH-KDEL部分按原顺序首位相连得到得到目的基因片段PE38-LHRH-KDEL进行序列测定。验证其基因序列的正确性后，扩增保存，并把目的基因片段，插入表达载体中，再把重组表达载体导入表达宿主菌中，经培养表达，纯化得到目的蛋白PE38-LHRH-KDEL，用MTT细胞测活法测定PE38-LHRH-KDEL的杀伤肿瘤细胞的活力。
此序列为PE38-LHRH-KDEL(序列16)
Gly-Gly-Ser-Leu-Ala-Ala-Leu-Thr-Ala-His-Gln-Ala-Cys-His-Leu-Pro-Leu-Glu-Thr-Phe-Thr-Arg-
His-Arg-Gln-Pro-Arg-Gly-Trp-Glu-Gln-Leu-Glu-Gln-Cys-Gly-Tyr-Pro-Val-Gln-Arg-Leu-Val-Ala-
Leu-Tyr-Leu-Ala-Ala-Arg-Leu-Ser-Trp-Asn-Gln-Val-Asp-Gln-Val-Ile-Arg-Asn-Ala-Leu-Ala-Ser-
Pro-Gly-Ser-Gly-Gly-Asp-Leu-Gly-Glu-Ala-Ile-Arg-Glu-Gln-Pro-Glu-Gln-Ala-Arg-Leu-Ala-Leu-
Thr-Leu-Ala-Ala-Ala-Glu-Ser-Glu-Arg-Phe-Val-Arg-Gln-Gly-Thr-Gly-Asn-Asp-Glu-Ala-Gly-Ala
-Ala-Asn-Gly-Pro-Ala-Asp-Ser-Gly-Asp-Ala-Leu-Leu-Glu-Arg-Asn-Tyr-Pro-Thr-Gly-Ala-Glu-
Phe-Leu-Gly-Asp-Gly-Gly-Asp-Val-Ser-Phe-Ser-Thr-Arg-Gly-Thr-Gln-Asn-Trp-Thr-Val-Glu-Arg-
Leu-Leu-Gln-Ala-His-Arg-Gln-Leu-Glu-Glu-Arg-Gly-Tyr-Val-Phe-Val-Gly-Tyr-His-Gly-Thr-Phe-
Leu-Glu-Ala-Ala-Gln-Ser-Ile-Val-Phe-Gly-Gly-Val-Arg-Ala-Arg-Ser-Gln-Asp-Leu-Asp-Ala-Ile-
Leu-Arg-Gly-Phe-Tyr-Ile-Ala-Gly-Asp-Pro-Ala-Leu-Ala-Tyr-Gly-Tyr-Ala-Gln-Asp-Gln-Glu-Pro-
Asp-Ala-Arg-Gly-Arg-Ile-Arg-Asn-Gly-Ala-Leu-Leu-Arg-Val-Tyr-Val-Pro-Arg-Ser-Ser-Leu-Pro-
Gly-Phe-Tyr-Arg-Thr-Ser-Leu-Thr-Leu-Ala-Ala-Pro-Glu-Ala-Ala-Gly-Glu-Val-Glu-Arg-Leu-Ile-
Gly-His-Pro-Leu-Pro-Leu-Arg-Leu-Asp-Ala-Ile-Thr-Gly-Pro-Glu-Glu-Glu-Gly-Gly-Arg-Leu-Glu-
Thr-Ile-Leu-Gly-Trp-Pro-Leu-Ala-Glu-Arg-Thr-Val-Val-Ile-Pro-Ser-Ala-Ile-Pro-Thr-Asp-Pro-Arg-
Asn-Val-Gly-Gly-Asp-Leu-Asp-Pro-Ser-Ser-Ile-Pro-Asp-Lys-Glu-Gln-Ala-Ile-Ser-Ala-Leu-Pro-
Asp-Tyr-Ala-Ser-Gln-Pro-Gly-Lys-Pro-Pro-Arg-Glu-Asp-Leu-Lys-Glu-His-Trp-Ser-Tyr-Gly-Leu-
Arg-Pro-Gly-Lys-Asp-Glu-Leu
实例十七
从pPE40-LHRH-KDEL载体用PCR方法把融合蛋白PE40部分缺失氨基酸254-279，365-380部分，取其氨基酸280-364和氨基酸381-613部分及LHRH-KDEL按原顺序首尾位相连得到PE35-LHRH-KDEL进行序列测定。验证其基因序列的正确性后，扩增保存，并把目的基因片段，插入表达载体中，再把重组表达载体导入表达宿主菌中，经培养表达，纯化得到目的蛋白PE35-LHRH-KDEL，用MTT细胞测活法测定PE35-LHRH-KDEL的杀伤肿瘤细胞的活力。
此序列为PE35-LHRH-KDEL(序列17)
Gly-Trp-Glu-Gln-Leu-Glu-Gln-Cys-Gly-Tyr-Pro-Val-Gln-Arg-Leu-Val-Ala-Leu-Tyr-Leu-Ala
-Ala-Arg-Leu-Ser-Trp-Asn-Gln-Val-Asp-Gln-Val-Ile-Arg-Asn-Ala-Leu-Ala-Ser-Pro-Gly-Ser-Gly-
Gly-Asp-Leu-Gly-Glu-Ala-Ile-Arg-Glu-Gln-Pro-Glu-Gln-Ala-Arg-Leu-Ala-Leu-Thr-Leu-Ala-Ala-
Ala-Glu-Ser-Glu-Arg-Phe-Val-Arg-Gln-Gly-Thr-Gly-Asn-Asp-Glu-Ala-Gly-Ala-Ala-Asn-Gly-Pro
-Ala-Asp-Ser-Gly-Asp-Ala-Leu-Leu-Glu-Arg-Asn-Tyr-Pro-Thr-Gly-Ala-Glu-Phe-Leu-Gly-Asp-
Gly-Gly-Asp-Val-Ser-Phe-Ser-Thr-Arg-Gly-Thr-Gln-Asn-Trp-Thr-Val-Glu-Arg-Leu-Leu-Gln-Ala-
His-Arg-Gln-Leu-Glu-Glu-Arg-Gly-Tyr-Val-Phe-Val-Gly-Tyr-His-Gly-Thr-Phe-Leu-Glu-Ala-Ala-
Gln-Ser-Ile-Val-Phe-Gly-Gly-Val-Arg-Ala-Arg-Ser-Gln-Asp-Leu-Asp-Ala-Ile-Trp-Arg-Gly-Phe-
Tyr-Ile-Ala-Gly-Asp-Pro-Ala-Leu-Ala-Tyr-Gly-Tyr-Ala-Gln-Asp-Gln-Glu-Pro-Asp-Ala-Arg-Gly-
Arg-Ile-Arg-Asn-Gly-Ala-Leu-Leu-Arg-Val-Tyr-Val-Pro-Arg-Ser-Ser-Leu-Pro-Gly-Phe-Tyr-Arg-
Thr-Ser-Leu-Thr-Leu-Ala-Ala-Pro-Glu-Ala-Ala-Gly-Glu-Val-Glu-Arg-Leu-Ile-Gly-His-Pro-Leu-
Pro-Leu-Arg-Leu-Asp-Ala-Ile-Thr-Gly-Pro-Glu-Glu-Glu-Gly-Gly-Arg-Leu-Glu-Thr-Ile-Leu-Gly-
Trp-Pro-Leu-Ala-Glu-Arg-Thr-Val-Val-Ile-Pro-Ser-Ala-Ile-Pro-Thr-Asp-Pro-Arg-Asn-Val-Gly-Gly
-Asp-Leu-Asp-Pro-Ser-Ser-Ile-Pro-Asp-Lys-Glu-Gln-Ala-Ile-Ser-Ala-Leu-Pro-Asp-Tyr-Ala-Ser-
Gln-Pro-Gly-Lys-Pro-Pro-Arg-Glu-Asp-Leu-Lys-Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-Lys-
Asp-Glu-Leu
实例十八
从pPE35-LHRH-KDEL载体上，用PCR方法把融合蛋白C末端4个氨基酸突变为REDLK，得到目的基因片段PE35-LHRH-REDLK进行序列测定。验证其基因序列的正确性后，扩增保存，并把目的基因片段，插入表达载体中，再把重组表达载体导入表达宿主菌中，经培养表达，纯化得到目的蛋白PE35-LHRH-REDLK，用MTT细胞测活法测定PE35-LHRH-REDLK的杀伤肿瘤细胞的活力。
此序列为PE35-LHRH-REDLK(序列18)
Gly-Trp-Glu-Gln-Leu-Glu-Gln-Cys-Gly-Tyr-Pro-Val-Gln-Arg-Leu-Val-Ala-Leu-Tyr-Leu-Ala
-Ala-Arg-Leu-Ser-Trp-Asn-Gln-Val-Asp-Gln-Val-Ile-Arg-Asn-Ala-Leu-Ala-Ser-Pro-Gly-Ser-Gly-
Gly-Asp-Leu-Gly-Glu-Ala-Ile-Arg-Glu-Gln-Pro-Glu-Gln-Ala-Arg-Leu-Ala-Leu-Thr-Leu-Ala-Ala-
Ala-Glu-Ser-Glu-Arg-Phe-Val-Arg-Gln-Gly-Thr-Gly-Asn-Asp-Glu-Ala-Gly-Ala-Ala-Asn-Gly-Pro
-Ala-Asp-Ser-Gly-Asp-Ala-Leu-Leu-Glu-Arg-Asn-Tyr-Pro-Thr-Gly-Ala-Glu-Phe-Leu-Gly-Asp-
Gly-Gly-Asp-Val-Ser-Phe-Ser-Thr-Arg-Gly-Thr-Gln-Asn-Trp-Thr-Val-Glu-Arg-Leu-Leu-Gln-Ala-
His-Arg-Gln-Leu-Glu-Glu-Arg-Gly-Tyr-Val-Phe-Val-Gly-Tyr-His-Gly-Thr-Phe-Leu-Glu-Ala-Ala-
Gln-Ser-Ile-Val-Phe-Gly-Gly-Val-Arg-Ala-Arg-Ser-Gln-Asp-Leu-Asp-Ala-Ile-Trp-Arg-Gly-Phe-
Tyr-Ile-Ala-Gly-Asp-Pro-Ala-Leu-Ala-Tyr-Gly-Tyr-Ala-Gln-Asp-Gln-Glu-Pro-Asp-Ala-Arg-Gly-
Arg-Ile-Arg-Asn-Gly-Ala-Leu-Leu-Arg-Val-Tyr-Val-Pro-Arg-Ser-Ser-Leu-Pro-Gly-Phe-Tyr-Arg-
Thr-Ser-Leu-Thr-Leu-Ala-Ala-Pro-Glu-Ala-Ala-Gly-Glu-Val-Glu-Arg-Leu-Ile-Gly-His-Pro-Leu-
Pro-Leu-Arg-Leu-Asp-Ala-Ile-Thr-Gly-Pro-Glu-Glu-Glu-Gly-Gly-Arg-Leu-Glu-Thr-Ile-Leu-Gly-
Trp-Pro-Leu-Ala-Glu-Arg-Thr-Val-Val-Ile-Pro-Ser-Ala-Ile-Pro-Thr-Asp-Pro-Arg-Asn-Val-Gly-Gly
-Asp-Leu-Asp-Pro-Ser-Ser-Ile-Pro-Asp-Lys-Glu-Gln-Ala-Ile-Ser-Ala-Leu-Pro-Asp-Tyr-Ala-Ser-
Gln-Pro-Gly-Lys-Pro-Pro-Arg-Glu-Asp-Leu-Lys-Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-Arg-
Glu-Asp-Leu-Lys
实例十九
从pPE40-LHRH-KDEL载体用PCR方法把融合蛋白LHRH部分N末端第6位氨基酸突变为Trp，得到目的基因片段PE40-LHRH(Trp6)-KDEL进行序列测定。验证其基因序列的正确性后，扩增保存，从该载体上切取下目的基因片段，插入表达载体中，再把重组表达载体导入表达宿主菌中，经培养表达，纯化得到目的蛋白PE40-LHRH(Trp6)-KDEL，用MTT细胞测活法测定PE40-LHRH(Trp6)-KDEL的杀伤肿瘤细胞的活力。
此序列为PE40-LHRH(Trp6)-KDEL(序列19)
Gly-Gly-Ser-Leu-Ala-Ala-Leu-Thr-Ala-His-Gln-Ala-Cys-His-Leu-Pro-Leu-Glu-Thr-Phe-Thr-Arg-
His-Arg-Gln-Pro-Arg-Gly-Trp-Glu-Gln-Leu-Glu-Gln-Cys-Gly-Tyr-Pro-Val-Gln-Arg-Leu-Val-Ala-
Leu-Tyr-Leu-Ala-Ala-Arg-Leu-Ser-Trp-Asn-Gln-Val-Asp-Gln-Val-Ile-Arg-Asn-Ala-Leu-Ala-Ser-
Pro-Gly-Ser-Gly-Gly-Asp-Leu-Gly-Glu-Ala-Ile-Arg-Glu-Gln-Pro-Glu-Gln-Ala-Arg-Leu-Ala-Leu-
Thr-Leu-Ala-Ala-Ala-Glu-Ser-Glu-Arg-Phe-Val-Arg-Gln-Gly-Thr-Gly-Asn-Asp-Glu-Ala-Gly-Ala
-Ala-Asn-Ala-Asp-Val-Val-Ser-Leu-Thr-Cys-Pro-Val-Ala-Ala-Gly-Glu-Cys-Ala-Gly-Pro-Ala-Asp-
Ser-Gly-Asp-Ala-Leu-Leu-Glu-Arg-Asn-Tyr-Pro-Thr-Gly-Ala-Glu-Phe-Leu-Gly-Asp-Gly-Gly-
Asp-Val-Ser-Phe-Ser-Thr-Arg-Gly-Thr-Gln-Asn-Trp-Thr-Val-Glu-Arg-Leu-Leu-Gln-Ala-His-Arg-
Gln-Leu-Glu-Glu-Arg-Gly-Tyr-Val-Phe-Val-Gly-Tyr-His-Gly-Thr-Phe-Leu-Glu-Ala-Ala-Gln-Ser-
Ile-Val-Phe-Gly-Gly-Val-Arg-Ala-Arg-Ser-Gln-Asp-Leu-Asp-Ala-Ile-Trp-Arg-Gly-Phe-Tyr-Ile-
Ala-Gly-Asp-Pro-Ala-Leu-Ala-Tyr-Gly-Tyr-Ala-Gln-Asp-Gln-Glu-Pro-Asp-Ala-Arg-Gly-Arg-Ile-
Arg-Asn-Gly-Ala-Leu-Leu-Arg-Val-Tyr-Val-Pro-Arg-Ser-Ser-Leu-Pro-Gly-Phe-Tyr-Arg-Thr-Ser-
Leu-Thr-Leu-Ala-Ala-Pro-Glu-Ala-Ala-Gly-Glu-Val-Glu-Arg-Leu-Ile-Gly-His-Pro-Leu-Pro-Leu-
Arg-Leu-Asp-Ala-Ile-Thr-Gly-Pro-Glu-Glu-Glu-Gly-Gly-Arg-Leu-Glu-Thr-Ile-Leu-Gly-Trp-Pro-
Leu-Ala-Glu-Arg-Thr-Val-Val-Ile-Pro-Ser-Ala-Ile-Pro-Thr-Asp-Pro-Arg-Asn-Val-Gly-Gly-Asp-
Leu-Asp-Pro-Ser-Ser-Ile-Pro-Asp-Lys-Glu-Gln-Ala-Ile-Ser-Ala-Leu-Pro-Asp-Tyr-Ala-Ser-Gln-Pro
-Gly-Lys-Pro-Pro-Arg-Glu-Asp-Leu-Lys-Glu-His-Trp-Ser-Tyr-Trp-Leu-Arg-Pro-Gly-Lys-Asp-
Glu-Leu
实例二十
从pPE35-LHRH-KDEL载体上，用PCR方法把融合蛋白LHRH部分N末端第6位氨基酸突变为Ala，得到目的基因片段PE35-LHRH(Ala6)-KDEL进行序列测定。验证其基因序列的正确性后，扩增保存，从该载体上切取下目的基因片段，插入表达载体中，再把重组表达载体导入表达宿主菌中，经培养表达，纯化得到目的蛋白PE35-LHRH(Ala6)-KDEL，用MTT细胞测活法测定PE35-LHRH(Ala6)-KDEL的杀伤肿瘤细胞的活力。
此序列为PE35-LHRH(Ala6)-KDEL(序列20)
Gly-Trp-Glu-Gln-Leu-Glu-Gln-Cys-Gly-Tyr-Pro-Val-Gln-Arg-Leu-Val-Ala-Leu-Tyr-Leu-Ala-Ala-
Arg-Leu-Ser-Trp-Asn-Gln-Val-Asp-Gln-Val-Ile-Arg-Asn-Ala-Leu-Ala-Ser-Pro-Gly-Ser-Gly-Gly-
Asp-Leu-Gly-Glu-Ala-Ile-Arg-Glu-Gln-Pro-Glu-Gln-Ala-Arg-Leu-Ala-Leu-Thr-Leu-Ala-Ala-Ala-
Glu-Ser-Glu-Arg-Phe-Val-Arg-Gln-Gly-Thr-Gly-Asn-Asp-Glu-Ala-Gly-Ala-Ala-Asn-Gly-Pro-Ala
-Asp-Ser-Gly-Asp-Ala-Leu-Leu-Glu-Arg-Asn-Tyr-Pro-Thr-Gly-Ala-Glu-Phe-Leu-Gly-Asp-Gly-
Gly-Asp-Val-Ser-Phe-Ser-Thr-Arg-Gly-Thr-Gln-Asn-Trp-Thr-Val-Glu-Arg-Leu-Leu-Gln-Ala-His-
Arg-Gln-Leu-Glu-Glu-Arg-Gly-Tyr-Val-Phe-Val-Gly-Tyr-His-Gly-Thr-Phe-Leu-Glu-Ala-Ala-Gln-
Ser-Ile-Val-Phe-Gly-Gly-Val-Arg-Ala-Arg-Ser-Gln-Asp-Leu-Asp-Ala-Ile-Trp-Arg-Gly-Phe-Tyr-Ile
-Ala-Gly-Asp-Pro-Ala-Leu-Ala-Tyr-Gly-Tyr-Ala-Gln-Asp-Gln-Glu-Pro-Asp-Ala-Arg-Gly-Arg-Ile
-Arg-Asn-Gly-Ala-Leu-Leu-Arg-Val-Tyr-Val-Pro-Arg-Ser-Ser-Leu-Pro-Gly-Phe-Tyr-Arg-Thr-Ser-
Leu-Thr-Leu-Ala-Ala-Pro-Glu-Ala-Ala-Gly-Glu-Val-Glu-Arg-Leu-Ile-Gly-His-Pro-Leu-Pro-Leu-
Arg-Leu-Asp-Ala-Ile-Thr-Gly-Pro-Glu-Glu-Glu-Gly-Gly-Arg-Leu-Glu-Thr-Ile-Leu-Gly-Trp-Pro-
Leu-Ala-Glu-Arg-Thr-Val-Val-Ile-Pro-Ser-Ala-Ile-Pro-Thr-Asp-Pro-Arg-Asn-Val-Gly-Gly-Asp-
Leu-Asp-Pro-Ser-Ser-Ile-Pro-Asp-Lys-Glu-Gln-Ala-Ile-Ser-Ala-Leu-Pro-Asp-Tyr-Ala-Ser-Gln-Pro
-Gly-Lys-Pro-Pro-Arg-Glu-Asp-Leu-Lys-Glu-His-Trp-Ser-Tyr-Ala-Leu-Arg-Pro-Gly-Lys-Asp-
Glu-Leu
实例二十一
从pPE40-LHRH(Trp6)-KDEL载体上，用PCR方法把融合蛋白C末端4个氨基酸突变为REDLK，得到目的基因片段PE40-LHRH(Trp6)-REDLK进行序列测定。验证其基因序列的正确性后，扩增保存，从该载体上切取下目的基因片段，插入表达载体中，再把重组表达载体导入表达宿主菌中，经培养表达，纯化得到目的蛋白PE40-LHRH(Trp6)-REDLK用MTT细胞测活法测定PE40-LHRH(Trp6)-REDLK的杀伤肿瘤细胞的活力。
此序列为PE40-LHRH(Trp6)-REDLK(序列21)
Gly-Gly-Ser-Leu-Ala-Ala-Leu-Thr-Ala-His-Gln-Ala-Cys-His-Leu-Pro-Leu-Glu-Thr-Phe-Thr-Arg-
His-Arg-Gln-Pro-Arg-Gly-Trp-Glu-Gln-Leu-Glu-Gln-Cys-Gly-Tyr-Pro-Val-Gln-Arg-Leu-Val-Ala-
Leu-Tyr-Leu-Ala-Ala-Arg-Leu-Ser-Trp-Asn-Gln-Val-Asp-Gln-Val-Ile-Arg-Asn-Ala-Leu-Ala-Ser-
Pro-Gly-Ser-Gly-Gly-Asp-Leu-Gly-Glu-Ala-Ile-Arg-Glu-Gln-Pro-Glu-Gln-Ala-Arg-Leu-Ala-Leu-
Thr-Leu-Ala-Ala-Ala-Glu-Ser-Glu-Arg-Phe-Val-Arg-Gln-Gly-Thr-Gly-Asn-Asp-Glu-Ala-Gly-Ala
-Ala-Asn-Ala-Asp-Val-Val-Ser-Leu-Thr-Cys-Pro-Val-Ala-Ala-Gly-Glu-Cys-Ala-Gly-Pro-Ala-Asp-
Ser-Gly-Asp-Ala-Leu-Leu-Glu-Arg-Asn-Tyr-Pro-Thr-Gly-Ala-Glu-Phe-Leu-Gly-Asp-Gly-Gly-
Asp-Val-Ser-Phe-Ser-Thr-Arg-Gly-Thr-Gln-Asn-Trp-Thr-Val-Glu-Arg-Leu-Leu-Gln-Ala-His-Arg-
Gln-Leu-Glu-Glu-Arg-Gly-Tyr-Val-Phe-Val-Gly-Tyr-His-Gly-Thr-Phe-Leu-Glu-Ala-Ala-Gln-Ser-
Ile-Val-Phe-Gly-Gly-Val-Arg-Ala-Arg-Ser-Gln-Asp-Leu-Asp-Ala-Ile-Trp-Arg-Gly-Phe-Tyr-Ile-
Ala-Gly-Asp-Pro-Ala-Leu-Ala-Tyr-Gly-Tyr-Ala-Gln-Asp-Gln-Glu-Pro-Asp-Ala-Arg-Gly-Arg-Ile-
Arg-Asn-Gly-Ala-Leu-Leu-Arg-Val-Tyr-Val-Pro-Arg-Ser-Ser-Leu-Pro-Gly-Phe-Tyr-Arg-Thr-Ser-
Leu-Thr-Leu-Ala-Ala-Pro-Glu-Ala-Ala-Gly-Glu-Val-Glu-Arg-Leu-Ile-Gly-His-Pro-Leu-Pro-Leu-
Arg-Leu-Asp-Ala-Ile-Thr-Gly-Pro-Glu-Glu-Glu-Gly-Gly-Arg-Leu-Glu-Thr-Ile-Leu-Gly-Trp-Pro-
Leu-Ala-Glu-Arg-Thr-Val-Val-Ile-Pro-Ser-Ala-Ile-Pro-Thr-Asp-Pro-Arg-Asn-Val-Gly-Gly-Asp-
Leu-Asp-Pro-Ser-Ser-Ile-Pro-Asp-Lys-Glu-Gln-Ala-Ile-Ser-Ala-Leu-Pro-Asp-Tyr-Ala-Ser-Gln-Pro
-Gly-Lys-Pro-Pro-Arg-Glu-Asp-Leu-Lys-Glu-His-Trp-Ser-Tyr-Trp-Leu-Arg-Pro-Gly-Arg-Glu-
Asp-Leu-Lys
实例二十二
从pPE35-LHRH(Ala6)-KDEL载体上，用PCR方法把融合蛋白C末端4个氨基酸突变为REDLK，得到目的基因片段PE35-LHRH(Ala6)-REDLK进行序列测定。验证其基因序列的正确性后，扩增保存，从该载体上切取下目的基因片段，插入表达载体中，再把重组表达载体导入表达宿主菌中，经培养表达，纯化得到目的蛋白PE35-LHRH(Ala6)-REDLK，用MTT细胞测活法测定PE35-LHRH(Ala6)-REDLK的杀伤肿瘤细胞的活力。
此序列为PE35-LHRH(Ala6)-REDLK(序列22)
Gly-Trp-Glu-Gln-Leu-Glu-Gln-Cys-Gly-Tyr-Pro-Val-Gln-Arg-Leu-Val-Ala-Leu-Tyr-Leu-Ala
-Ala-Arg-Leu-Ser-Trp-Asn-Gln-Val-Asp-Gln-Val-Ile-Arg-Asn-Ala-Leu-Ala-Ser-Pro-Gly-Ser-Gly-
Gly-Asp-Leu-Gly-Glu-Ala-Ile-Arg-Glu-Gln-Pro-Glu-Gln-Ala-Arg-Leu-Ala-Leu-Thr-Leu-Ala-Ala-
Ala-Glu-Ser-Glu-Arg-Phe-Val-Arg-Glr-Gly-Thr-Gly-Asn-Asp-Glu-Ala-Gly-Ala-Ala-Asn-Gly-Pro
-Ala-Asp-Ser-Gly-Asp-Ala-Leu-Leu-Glu-Arg-Asn-Tyr-Pro-Thr-Gly-Ala-Glu-Phe-Leu-Gly-Asp-
Gly-Gly-Asp-Val-Ser-Phe-Ser-Thr-Arg-Gly-Thr-Gln-Asn-Trp-Thr-Val-Glu-Arg-Leu-Leu-Gln-Ala-
His-Arg-Gln-Leu-Glu-Glu-Arg-Gly-Tyr-Val-Phe-Val-Gly-Tyr-His-Gly-Thr-Phe-Leu-Glu-Ala-Ala-
Gln-Ser-Ile-Val-Phe-Gly-Gly-Val-Arg-Ala-Arg-Ser-Gln-Asp-Leu-Asp-Ala-Ile-Trp-Arg-Gly-Phe-
Tyr-Ile-Ala-Gly-Asp-Pro-Ala-Leu-Ala-Tyr-Gly-Tyr-Ala-Gln-Asp-Gln-Glu-Pro-Asp-Ala-Arg-Gly-
Arg-Ile-Arg-Asn-Gly-Ala-Leu-Leu-Arg-Val-Tyr-Val-Pro-Arg-Ser-Ser-Leu-Pro-Gly-Phe-Tyr-Arg-
Thr-Ser-Leu-Thr-Leu-Ala-Ala-Pro-Glu-Ala-Ala-Gly-Glu-Val-Glu-Arg-Leu-Ile-Gly-His-Pro-Leu-
Pro-Leu-Arg-Leu-Asp-Ala-Ile-Thr-Gly-Pro-Glu-Glu-Glu-Gly-Gly-Arg-Leu-Glu-Thr-Ile-Leu-Gly-
Trp-Pro-Leu-Ala-Glu-Arg-Thr-Val-Val-Ile-Pro-Ser-Ala-Ile-Pro-Thr-Asp-Pro-Arg-Asn-Val-Gly-Gly
-Asp-Leu-Asp-Pro-Ser-Ser-Ile-Pro-Asp-Lys-Glu-Gln-Ala-Ile-Ser-Ala-Leu-Pro-Asp-Tyr-Ala-Ser-
Gln-Pro-Gly-Lys-Pro-Pro-Arg-Glu-Asp-Leu-Lys-Glu-His-Trp-Ser-Tyr-Ala-Leu-Arg-Pro-Gly-Arg-
Glu-Asp-Leu-Lys
实例二十三
从pLHRH-PE40载体上，用PCR方法把融合蛋白PE40部分C末端5个氨基酸突变为KDELKDELKDEL，得到目的基因片段LHRH-PE40-KDELKDELKDEL进行序列测定。验证其基因序列的正确性后，扩增保存，从该载体上切取下目的基因片段，插入表达载体中，再把重组表达载体导入表达宿主菌中，经培养表达，纯化得到目的蛋白LHRH-PE40-KDELKDELKDEL，用MTT细胞测活法测定LHRH-PE40-KDELKDELKDEL的杀伤肿瘤细胞的活力。
此序列为LHRH-PE40-KDELKDELKDEL(序列23)
Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-Gly-Gly-Ser-Leu-Ala-Ala-Leu-Thr-Ala-His-Gln-Ala-
Cys-His-Leu-Pro-Leu-Glu-Thr-Phe-Thr-Arg-His-Arg-Gln-Pro-Arg-Gly-Trp-Glu-Gln-Leu-Glu-Gln
-Cys-Gly-Tyr-Pro-Val-Gln-Arg-Leu-Val-Ala-Leu-Tyr-Leu-Ala-Ala-Arg-Leu-Ser-Trp-Asn-Gln-Val-
Asp-Gln-Val-Ile-Arg-Asn-Ala-Leu-Ala-Ser-Pro-Gly-Ser-Gly-Gly-Asp-Leu-Gly-Glu-Ala-Ile-Arg-
Glu-Gln-Pro-Glu-Gln-Ala-Arg-Leu-Ala-Leu-Thr-Leu-Ala-Ala-Ala-Glu-Ser-Glu-Arg-Phe-Val-Arg-
Gln-Gly-Thr-Gly-Asn-Asp-Glu-Ala-Gly-Ala-Ala-Asn-Ala-Asp-Val-Val-Ser-Leu-Thr-Cys-Pro-Val-
Ala-Ala-Gly-Glu-Cys-Ala-Gly-Pro-Ala-Asp-Ser-Gly-Asp-Ala-Leu-Leu-Glu-Arg-Asn-Tyr-Pro-Thr
-Gly-Ala-Glu-Phe-Leu-Gly-Asp-Gly-Gly-Asp-Val-Ser-Phe-Ser-Thr-Arg-Gly-Thr-Gln-Asn-Trp-
Thr-Val-Glu-Arg-Leu-Leu-Gln-Ala-His-Arg-Gln-Leu-Glu-Glu-Arg-Gly-Tyr-Val-Phe-Val-Gly-Tyr-
His-Gly-Thr-Phe-Leu-Glu-Ala-Ala-Gln-Ser-Ile-Val-Phe-Gly-Gly-Val-Arg-Ala-Arg-Ser-Gln-Asp-
Leu-Asp-Ala-Ile-Trp-Arg-Gly-Phe-Tyr-Ile-Ala-Gly-Asp-Pro-Ala-Leu-Ala-Tyr-Gly-Tyr-Ala-Gln-
Asp-Gln-Glu-Pro-Asp-Ala-Arg-Gly-Arg-Ile-Arg-Asn-Gly-Ala-Leu-Leu-Arg-Val-Tyr-Val-Pro-Arg-
Ser-Ser-Leu-Pro-Gly-Phe-Tyr-Arg-Thr-Ser-Leu-Thr-Leu-Ala-Ala-Pro-Glu-Ala-Ala-Gly-Glu-Val-
Glu-Arg-Leu-Ile-Gly-His-Pro-Leu-Pro-Leu-Arg-Leu-Asp-Ala-Ile-Thr-Gly-Pro-Glu-Glu-Glu-Gly-
Gly-Arg-Leu-Glu-Thr-Ile-Leu-Gly-Trp-Pro-Leu-Ala-Glu-Arg-Thr-Val-Val-Ile-Pro-Ser-Ala-Ile-Pro-
Thr-Asp-Pro-Arg-Asn-Val-Gly-Gly-Asp-Leu-Asp-Pro-Ser-Ser-Ile-Pro-Asp-Lys-Glu-Gln-Ala-Ile-
Ser-Ala-Leu-Pro-Asp-Tyr-Ala-Ser-Gln-Pro-Gly-Lys-Pro-Pro-Lys-Asp-Glu-Leu-Lys-Asp-Glu-
Leu-Lys-Asp-Glu-Leu
实例二十四
从pLHRH-PE40-KDELKDELKDEL载体上，用PCR方法把融合蛋白LHRH部分N末端第6位氨基酸突变为Trp，得到目的基因片段LHRH(Trp6)-PE40-KDELKDELKDEL进行序列测定。验证其基因序列的正确性后，扩增保存，从该载体上切取下目的基因片段，插入表达载体中，再把重组表达载体导入表达宿主菌中，经培养表达，纯化得到目的蛋白LHRH(Trp6)-PE40-KDELKDELKDEL，用MTT细胞测活法测定LHRH(Trp6)-PE40-KDELKDELKDEL的杀伤肿瘤细胞的活力。
此序列为LHRH(Trp6)-PE40-KDELKDELKDEL(序列24)
Glu-His-Trp-Ser-Tyr-Trp-Leu-Arg-Pro-Gly-Gly-Gly-Ser-Leu-Ala-Ala-Leu-Thr-Ala-His-Gln-Ala-
Cys-His-Leu-Pro-Leu-Glu-Thr-Phe-Thr-Arg-His-Arg-Gln-Pro-Arg-Gly-Trp-Glu-Gln-Leu-Glu-Gln
-Cys-Gly-Tyr-Pro-Val-Gln-Arg-Leu-Val-Ala-Leu-Tyr-Leu-Ala-Ala-Arg-Leu-Ser-Trp-Asn-Gln-Val-
Asp-Gln-Val-Ile-Arg-Asn-Ala-Leu-Ala-Ser-Pro-Gly-Ser-Gly-Gly-Asp-Leu-Gly-Glu-Ala-Ile-Arg-
Glu-Gln-Pro-Glu-Gln-Ala-Arg-Leu-Ala-Leu-Thr-Leu-Ala-Ala-Ala-Glu-Ser-Glu-Arg-Phe-Val-Arg-
Gln-Gly-Thr-Gly-Asn-Asp-Glu-Ala-Gly-Ala-Ala-Asn-Ala-Asp-Val-Val-Ser-Leu-Thr-Cys-Pro-Val-
Ala-Ala-Gly-Glu-Cys-Ala-Gly-Pro-Ala-Asp-Ser-Gly-Asp-Ala-Leu-Leu-Glu-Arg-Asn-Tyr-Pro-Thr
-Gly-Ala-Glu-Phe-Leu-Gly-Asp-Gly-Gly-Asp-Val-Ser-Phe-Ser-Thr-Arg-Gly-Thr-Gln-Asn-Trp-
Thr-Val-Glu-Arg-Leu-Leu-Gln-Ala-His-Arg-Gln-Leu-Glu-Glu-Arg-Gly-Tyr-Val-Phe-Val-Gly-Tyr-
His-Gly-Thr-Phe-Leu-Glu-Ala-Ala-Gln-Ser-Ile-Val-Phe-Gly-Gly-Val-Arg-Ala-Arg-Ser-Gln-Asp-
Leu-Asp-Ala-Ile-Trp-Arg-Gly-Phe-Tyr-Ile-Ala-Gly-Asp-Pro-Ala-Leu-Ala-Tyr-Gly-Tyr-Ala-Gln-
Asp-Gln-Glu-Pro-Asp-Ala-Arg-Gly-Arg-Ile-Arg-Asn-Gly-Ala-Leu-Leu-Arg-Val-Tyr-Val-Pro-Arg-
Ser-Ser-Leu-Pro-Gly-Phe-Tyr-Arg-Thr-Ser-Leu-Thr-Leu-Ala-Ala-Pro-Glu-Ala-Ala-Gly-Glu-Val-
Glu-Arg-Leu-Ile-Gly-His-Pro-Leu-Pro-Leu-Arg-Leu-Asp-Ala-Ile-Thr-Gly-Pro-Glu-Glu-Glu-Gly-
Gly-Arg-Leu-Glu-Thr-Ile-Leu-Gly-Trp-Pro-Leu-Ala-Glu-Arg-Thr-Val-Val-Ile-Pro-Ser-Ala-Ile-Pro-
Thr-Asp-Pro-Arg-Asn-Val-Gly-Gly-Asp-Leu-Asp-Pro-Ser-Ser-Ile-Pro-Asp-Lys-Glu-Gln-Ala-Ile-
Ser-Ala-Leu-Pro-Asp-Tyr-Ala-Ser-Gln-Pro-Gly-Lys-Pro-Pro-Lys-Asp-Glu-Leu-Lys-Asp-Glu-Leu-
Lys-Asp-Glu-Leu
实例二十五
从pLHRH-PE35载体上，用PCR方法把融合蛋白PE35部分C末端5个氨基酸突变为KDELKDELKDEL，得到目的基因片段LHRH-PE35-KDELKDELKDEL进行序列测定。验证其基因序列的正确性后，扩增保存，从该载体上切取下目的基因片段，插入表达载体中，再把重组表达载体导入表达宿主菌中，经培养表达，纯化得到目的蛋白LHRH-PE35-KDELKDELKDEL，用MTT细胞测活法测定LHRH-PE35-KDELKDELKDEL的杀伤肿瘤细胞的活力。
此序列为LHRH-PE35-KDELKDELKDEL(序列25)
Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-Gly-Trp-Glu-Gln-Leu-Glu-Gln-Cys-Gly-Tyr-Pro-Val-
Gln-Arg-Leu-Val-Ala-Leu-Tyr-Leu-Ala-Ala-Arg-Leu-Ser-Trp-Asn-Gln-Val-Asp-Gln-Val-Ile-Arg-
Asn-Ala-Leu-Ala-Ser-Pro-Gly-Ser-Gly-Gly-Asp-Leu-Gly-Glu-Ala-Ile-Arg-Glu-Gln-Pro-Glu-Gln-
Ala-Arg-Leu-Ala-Leu-Thr-Leu-Ala-Ala-Ala-Glu-Ser-Glu-Arg-Phe-Val-Arg-Gln-Gly-Thr-Gly-Asn
-Asp-Glu-Ala-Gly-Ala-Ala-Asn-Gly-Pro-Ala-Asp-Ser-Gly-Asp-Ala-Leu-Leu-Glu-Arg-Asn-Tyr-
Pro-Thr-Gly-Ala-Glu-Phe-Leu-Gly-Asp-Gly-Gly-Asp-Val-Ser-Phe-Ser-Thr-Arg-Gly-Thr-Gln-Asn-
Trp-Thr-Val-Glu-Arg-Leu-Leu-Gln-Ala-His-Arg-Gln-Leu-Glu-Glu-Arg-Gly-Tyr-Val-Phe-Val-Gly-
Tyr-His-Gly-Thr-Phe-Leu-Glu-Ala-Ala-Gln-Ser-Ile-Val-Phe-Gly-Gly-Val-Arg-Ala-Arg-Ser-Gln-
Asp-Leu-Asp-Ala-Ile-Trp-Arg-Gly-Phe-Tyr-Ile-Ala-Gly-Asp-Pro-Ala-Leu-Ala-Tyr-Gly-Tyr-Ala-
Gln-Asp-Gln-Glu-Pro-Asp-Ala-Arg-Gly-Arg-Ile-Arg-Asn-Gly-Ala-Leu-Leu-Arg-Val-Tyr-Val-Pro-
Arg-Ser-Ser-Leu-Pro-Gly-Phe-Tyr-Arg-Thr-Ser-Leu-Thr-Leu-Ala-Ala-Pro-Glu-Ala-Ala-Gly-Glu-
Val-Glu-Arg-Leu-Ile-Gly-His-Pro-Leu-Pro-Leu-Arg-Leu-Asp-Ala-Ile-Thr-Gly-Pro-Glu-Glu-Glu-
Gly-Gly-Arg-Leu-Glu-Thr-Ile-Leu-Gly-Trp-Pro-Leu-Ala-Glu-Arg-Thr-Val-Val-Ile-Pro-Ser-Ala-Ile
-Pro-Thr-Asp-Pro-Arg-Asn-Val-Gly-Gly-Asp-Leu-Asp-Pro-Ser-Ser-Ile-Pro-Asp-Lys-Glu-Gln-Ala-
Ile-Ser-Ala-Leu-Pro-Asp-Tyr-Ala-Ser-Gln-Pro-Gly-Lys-Pro-Pro-Lys-Asp-Glu-Leu-Lys-Asp-Glu-
Leu-Lys-Asp-Glu-Leu
实例二十六
从pLHRH-PE35-KDELKDELKDEL载体用PCR方法把融合蛋白LHRH部分N末端第6个氨基酸突变为Ala，得到目的基因片段LHRH(Ala6)-PE35-KDELKDELKDEL进行序列测定。验证其基因序列的正确性后，扩增保存，从该载体上切取下目的基因片段，插入表达载体中，再把重组表达载体导入表达宿主菌中，经培养表达，纯化得到目的蛋白LHRH(Ala6)-PE35-KDELKDELKDEL，用MTT细胞测活法测定LHRH(Ala6)-PE35-KDELKDELKDEL的杀伤肿瘤细胞的活力。
此序列为LHRH(Ala6)-PE35-KDELKDELKDEL(序列26)
Glu-His-Trp-Ser-Tyr-Ala-Leu-Arg-Pro-Gly-Gly-Trp-Glu-Gln-Leu-Glu-Gln-Cys-Gly-Tyr-Pro-Val-
Gln-Arg-Leu-Val-Ala-Leu-Tyr-Leu-Ala-Ala-Arg-Leu-Ser-Trp-Asn-Gln-Val-Asp-Gln-Val-Ile-Arg-
Asn-Ala-Leu-Ala-Ser-Pro-Gly-Ser-Gly-Gly-Asp-Leu-Gly-Glu-Ala-Ile-Arg-Glu-Gln-Pro-Glu-Gln-
Ala-Arg-Leu-Ala-Leu-Thr-Leu-Ala-Ala-Ala-Glu-Ser-Glu-Arg-Phe-Val-Arg-Gln-Gly-Thr-Gly-Asn
-Asp-Glu-Ala-Gly-Ala-Ala-Asn-Gly-Pro-Ala-Asp-Ser-Gly-Asp-Ala-Leu-Leu-Glu-Arg-Asn-Tyr-
Pro-Thr-Gly-Ala-Glu-Phe-Leu-Gly-Asp-Gly-Gly-Asp-Val-Ser-Phe-Ser-Thr-Arg-Gly-Thr-Gln-Asn-
Trp-Thr-Val-Glu-Arg-Leu-Leu-Gln-Ala-His-Arg-Gln-Leu-Glu-Glu-Arg-Gly-Tyr-Val-Phe-Val-Gly-
Tyr-His-Gly-Thr-Phe-Leu-Glu-Ala-Ala-Gln-Ser-Ile-Val-Phe-Gly-Gly-Val-Arg-Ala-Arg-Ser-Gln-
Asp-Leu-Asp-Ala-Ile-Trp-Arg-Gly-Phe-Tyr-Ile-Ala-Gly-Asp-Pro-Ala-Leu-Ala-Tyr-Gly-Tyr-Ala-
Gln-Asp-Gln-Glu-Pro-Asp-Ala-Arg-Gly-Arg-Ile-Arg-Asn-Gly-Ala-Leu-Leu-Arg-Val-Tyr-Val-Pro-
Arg-Ser-Ser-Leu-Pro-Gly-Phe-Tyr-Arg-Thr-Ser-Leu-Thr-Leu-Ala-Ala-Pro-Glu-Ala-Ala-Gly-Glu-
Val-Glu-Arg-Leu-Ile-Gly-His-Pro-Leu-Pro-Leu-Arg-Leu-Asp-Ala-Ile-Thr-Gly-Pro-Glu-Glu-Glu-
Gly-Gly-Arg-Leu-Glu-Thr-Ile-Leu-Gly-Trp-Pro-Leu-Ala-Glu-Arg-Thr-Val-Val-Ile-Pro-Ser-Ala-Ile
-Pro-Thr-Asp-Pro-Arg-Asn-Val-Gly-Gly-Asp-Leu-Asp-Pro-Ser-Ser-Ile-Pro-Asp-Lys-Glu-Gln-Ala-
Ile-Ser-Ala-Leu-Pro-Asp-Tyr-Ala-Ser-Gln-Pro-Gly-Lys-Pro-Pro-Lys-Asp-Glu-Leu-Lys-Asp-Glu-
Leu-Lys-Asp-Glu-Leu
实例二十七
用MTT比色法测定细胞增殖中活细胞的数目，以此确定融和蛋白的杀伤各种肿瘤细胞的活力，所选的肿瘤细胞包括SPC(人肺腺癌细胞)、HL60(人白血病细胞)、MGC(人胃癌细胞)、PC-3(人前列腺癌细胞)和MCF-7(人乳腺癌细胞)。取96孔细胞培养板3块，每孔接种细胞数为1×104、每块培养板每种细胞重复接种20个孔，37℃、5％ CO2条件下培养，然后分别加入等比稀释融和蛋白样品，接着培养24，48及72h后，每孔加5g/L MTT 20μl再培养4h，然后去上清液加入二甲亚砜200μl，将培养板放在微量振荡器上振荡10min，待MTT被细胞还原形成甲颗粒完全溶解后，立即用酶标仪、波长570nm测定其吸光度，然后通过标准计算公式计算出目的蛋白活性。上述实验重复3次，计算其平均IC50值。
结果：见表1，以LHRH-PE40为对照，实例1-7中融和蛋白突变体是单独对PE40部分进行的突变，活性大约提高了5-20倍；实例8中的融和蛋白突变体是单独对LHRH部分进行的突变，活性提高了10倍；实例9-14中的融和蛋白突变体是结合了LHRH和PE40部分二者的突变体，活性提高了50-1000倍；实例15-22中的融和蛋白突变体活性提高了20-500倍；实例22-26中的融和蛋白突变体活性提高了100-800倍。
表1.实例中的融合蛋白细胞活性
 序号  细胞IC50融合蛋白 SPC(ug/mL)  HL60(ug/mL)  MGC(ug/mL)  PC-3(ug/mL)  MCF-7(ug/mL)
 0  LHRH-PE40 28.2  19.8  52.1  2.75  1.48
 1  LHRH-PE40(KDEL) 6.8  15.4  20.1  0.58  0.54
 2  LHRH-PE40(RQEL) 10.7  19.1  16.7  1.3  0.48
 3  LHRH-PE40(KEEL) 8.2  22.4  21.2  0.86  0.62
 4  LHRH-PE40(REEL)  7.6  15.7  18.3  1.2  0.37
 5  LHRH-PE38  3.2  2.4  4.3  0.25  0.22
 6  LHRH-PE35  2.1  1.3  6.8  0.18  0.25
 7  LHRH-PE35(KDEL)  1.5  1.4  2.6  0.14  0.12
 8  LHRH(Trp6)-PE40  3.1  2.1  5.3  0.28  0.16
 9  LHRH(Trp6)-PE40(KDEL)  1.1  1.8  1.2  0.061  0.032
 10  LHRH(Trp6)-PE38  0.24  0.28  0.66  0.019  0.022
 11  LHRH(Trp6)-PE38(KDEL)  0.062  0.055  0.17  0.0052  0.0038
 12  LHRH(Ala6)-PE35  0.18  0.22  0.74  0.057  0.041
 13  LHRH(Ala6)-PE35(KDEL)  0.022  0.025  0.48  0.0013  0.0021
 14  LHRH(Ala6)-PE35(RQEL)  0.046  0.032  0.28  0.0037  0.0018
 15  PE40-LHRH-KDEL  2.6  1.4  3.9  0.17  0.048
 16  PE38-LHRH-KDEL  2.2  0.65  2.8  0.095  0.041
 17  PE35-LHRH-KDEL  3.41  0.66  2.2  0.11  0.026
 18  PE35-LHRH-REDLK  2.7  0.42  1.9  0.074  0.023
 19  PE40-LHRH(Trp6)-KDEL  0.12  0.077  0.22  0.012  0.0052
 20  PE35-LHRH(Ala6)-KDEL  0.074  0.041  0.16  0.0071  0.0022
 21  PE40-LHRH(Trp6)-REDLK  0.33  0.35  0.69  0.26  0.21
 22  PE35-LHRH(Ala6)-REDLK  0.062  0.081  0.14  0.044  0.022
 23  LHRH-PE40(KDEL)3  0.41  0.16  0.32  0.35  0.16
 24  LHRH(Trp6)-PE40(KDEL)3  0.085  0.055  0.062  0.077  0.043
 25  LHRH(Ala6)-PE35(KDEL)3  0.022  0.031  0.064  0.0018  0.0011
五.序列表
(1)序列1
Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-Gly-Gly-Ser-Leu-Ala-Ala-Leu-Thr-Ala-His-Gln-Ala-
Cys-His-Leu-Pro-Leu-Glu-Thr-Phe-Thr-Arg-His-Arg-Gln-Pro-Arg-Gly-Trp-Glu-Gln-Leu-Glu-Gln
-Cys-Gly-Tyr-Pro-Val-Gln-Arg-Leu-Val-Ala-Leu-Tyr-Leu-Ala-Ala-Arg-Leu-Ser-Trp-Asn-Gln-Val-
Asp-Gln-Val-Ile-Arg-Asn-Ala-Leu-Ala-Ser-Pro-Gly-Ser-Gly-Gly-Asp-Leu-Gly-Glu-Ala-Ile-Arg-
Glu-Gln-Pro-Glu-Gln-Ala-Arg-Leu-Ala-Leu-Thr-Leu-Ala-Ala-Ala-Glu-Ser-Glu-Arg-Phe-Val-Arg-
Gln-Gly-Thr-Gly-Asn-Asp-Glu-Ala-Gly-Ala-Ala-Asn-Ala-Asp-Val-Val-Ser-Leu-Thr-Cys-Pro-Val-
Ala-Ala-Gly-Glu-Cys-Ala-Gly-Pro-Ala-Asp-Ser-Gly-Asp-Ala-Leu-Leu-Glu-Arg-Asn-Tyr-Pro-Thr
-Gly-Ala-Glu-Phe-Leu-Gly-Asp-Gly-Gly-Asp-Val-Ser-Phe-Ser-Thr-Arg-Gly-Thr-Gln-Asn-Trp-Th
r-Val-Glu-Arg-Leu-Leu-Gln-Ala-His-Arg-Gln-Leu-Glu-Glu-Arg-Gly-Tyr-Val-Phe-Val-Gly-Tyr-His
-Gly-Thr-Phe-Leu-Glu-Ala-Ala-Gln-Ser-Ile-Val-Phe-Gly-Gly-Val-Arg-Ala-Arg-Ser-Gln-Asp-Leu-
Asp-Ala-Ile-Trp-Arg-Gly-Phe-Tyr-Ile-Ala-Gly-Asp-Pro-Ala-Leu-Ala-Tyr-Gly-Tyr-Ala-Gln-Asp-
Gln-Glu-Pro-Asp-Ala-Arg-Gly-Arg-Ile-Arg-Asn-Gly-Ala-Leu-Leu-Arg-Val-Tyr-Val-Pro-Arg-Ser-
Ser-Leu-Pro-Gly-Phe-Tyr-Arg-Thr-Ser-Leu-Thr-Leu-Ala-Ala-Pro-Glu-Ala-Ala-Gly-Glu-Val-Glu-
Arg-Leu-Ile-Gly-His-Pro-Leu-Pro-Leu-Arg-Leu-Asp-Ala-Ile-Thr-Gly-Pro-Glu-Glu-Glu-Gly-Gly-
Arg-Leu-Glu-Thr-Ile-Leu-Gly-Trp-Pro-Leu-Ala-Glu-Arg-Thr-Val-Val-Ile-Pro-Ser-Ala-Ile-Pro-Thr-
Asp-Pro-Arg-Asn-Val-Gly-Gly-Asp-Leu-Asp-Pro-Ser-Ser-Ile-Pro-Asp-Lys-Glu-Gln-Ala-Ile-Ser-
Ala-Leu-Pro-Asp-Tyr-Ala-Ser-Gln-Pro-Gly-Lys-Pro-Pro-(Lys-Asp-Glu-Leu)n
说明：该序列的N末端第6位Gly可替换成为Ala、Trp或Leu；该序列的C末端第3位Asp可替换成Gln或Glu；该序列C末端第4位Lys可替换成Arg。n小于等于5大于等于1。并且该序列C末端的氨基酸-(Lys-Asp-Glu-Leu)n可替换成-(Arg-Glu-Asp-Leu-Lys)n，n小于等于5大于等于1。
(2)序列2
Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-Gly-Gly-Ser-Leu-Ala-Ala-Leu-Thr-Ala-His-Gln-Ala-
Cys-His-Leu-Pro-Leu-Glu-Thr-Phe-Thr-Arg-His-Arg-Gln-Pro-Arg-Gly-Trp-Glu-Gln-Leu-Glu-Gln
-Cys-Gly-Tyr-Pro-Val-Gln-Arg-Leu-Val-Ala-Leu-Tyr-Leu-Ala-Ala-Arg-Leu-Ser-Trp-Asn-Gln-Val-
Asp-Gln-Val-Ile-Arg-Asn-Ala-Leu-Ala-Ser-Pro-Gly-Ser-Gly-Gly-Asp-Leu-Gly-Glu-Ala-Ile-Arg-
Glu-Gln-Pro-Glu-Gln-Ala-Arg-Leu-Ala-Leu-Thr-Leu-Ala-Ala-Ala-Glu-Ser-Glu-Arg-Phe-Val-Arg-
Gln-Gly-Thr-Gly-Asn-Asp-Glu-Ala-Gly-Ala-Ala-Asn-Gly-Pro-Ala-Asp-Ser-Gly-Asp-Ala-Leu-
Leu-Glu-Arg-Asn-Tyr-Pro-Thr-Gly-Ala-Glu-Phe-Leu-Gly-Asp-Gly-Gly-Asp-Val-Ser-Phe-Ser-Thr-
Arg-Gly-Thr-Gln-Asn-Trp-Thr-Val-Glu-Arg-Leu-Leu-Gln-Ala-His-Arg-Gln-Leu-Glu-Glu-Arg-Gly
-Tyr-Val-Phe-Val-Gly-Tyr-His-Gly-Thr-Phe-Leu-Glu-Ala-Ala-Gln-Ser-Ile-Val-Phe-Gly-Gly-Val-
Arg-Ala-Arg-Ser-Gln-Asp-Leu-Asp-Ala-Ile-Trp-Arg-Gly-Phe-Tyr-Ile-Ala-Gly-Asp-Pro-Ala-Leu-
Ala-Tyr-Gly-Tyr-Ala-Gln-Asp-Gln-Glu-Pro-Asp-Ala-Arg-Gly-Arg-Ile-Arg-Asn-Gly-Ala-Leu-Leu-
Arg-Val-Tyr-Val-Pro-Arg-Ser-Ser-Leu-Pro-Gly-Phe-Tyr-Arg-Thr-Ser-Leu-Thr-Leu-Ala-Ala-Pro-
Glu-Ala-Ala-Gly-Glu-Val-Glu-Arg-Leu-Ile-Gly-His-Pro-Leu-Pro-Leu-Arg-Leu-Asp-Ala-Ile-Thr-
Gly-Pro-Glu-Glu-Glu-Gly-Gly-Arg-Leu-Glu-Thr-Ile-Leu-Gly-Trp-Pro-Leu-Ala-Glu-Arg-Thr-Val-
Val-Ile-Pro-Ser-Ala-Ile-Pro-Thr-Asp-Pro-Arg-Asn-Val-Gly-Gly-Asp-Leu-Asp-Pro-Ser-Ser-Ile-Pro-
Asp-Lys-Glu-Gln-Ala-Ile-Ser-Ala-Leu-Pro-Asp-Tyr-Ala-Ser-Gln-Pro-Gly-Lys-Pro-Pro-(Lys-Asp-
Glu-Leu)n
说明：该序列的N末端第6位Gly可替换成为Ala、Trp或Leu；该序列的C末端第3位Asp可替换成Gln或Glu；该序列C末端第4位Lys可替换成Arg。n小于等于5大于等于1。并且该序列C末端的氨基酸-(Lys-Asp-Glu-Leu)n可替换成-(Arg-Glu-Asp-Leu-Lys)n，n小于等于5大于等于1。
(3)序列3
Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-Gly-Trp-Glu-Gln-Leu-Glu-Gln-Cys-Gly-Tyr-Pro-Val-
Gln-Arg-Leu-Val-Ala-Leu-Tyr-Leu-Ala-Ala-Arg-Leu-Ser-Trp-Asn-Gln-Val-Asp-Gln-Val-Ile-Arg-
Asn-Ala-Leu-Ala-Ser-Pro-Gly-Ser-Gly-Gly-Asp-Leu-Gly-Glu-Ala-Ile-Arg-Glu-Gln-Pro-Glu-Gln-
Ala-Arg-Leu-Ala-Leu-Thr-Leu-Ala-Ala-Ala-Glu-Ser-Glu-Arg-Phe-Val-Arg-Gln-Gly-Thr-Gly-Asn
-Asp-Glu-Ala-Gly-Ala-Ala-Asn-Gly-Pro-Ala-Asp-Ser-Gly-Asp-Ala-Leu-Leu-Glu-Arg-Asn-Tyr-
Pro-Thr-Gly-Ala-Glu-Phe-Leu-Gly-Asp-Gly-Gly-Asp-Val-Ser-Phe-Ser-Thr-Arg-Gly-Thr-Gln-Asn-
Trp-Thr-Val-Glu-Arg-Leu-Leu-Gln-Ala-His-Arg-Gln-Leu-Glu-Glu-Arg-Gly-Tyr-Val-Phe-Val-Gly-
Tyr-His-Gly-Thr-Phe-Leu-Glu-Ala-Ala-Gln-Ser-Ile-Val-Phe-Gly-Gly-Val-Arg-Ala-Arg-Ser-Gln-
Asp-Leu-Asp-Ala-Ile-Trp-Arg-Gly-Phe-Tyr-Ile-Ala-Gly-Asp-Pro-Ala-Leu-Ala-Tyr-Gly-Tyr-Ala-
Gln-Asp-Gln-Glu-Pro-Asp-Ala-Arg-Gly-Arg-Ile-Arg-Asn-Gly-Ala-Leu-Leu-Arg-Val-Tyr-Val-Pro-
Arg-Ser-Ser-Leu-Pro-Gly-Phe-Tyr-Arg-Thr-Ser-Leu-Thr-Leu-Ala-Ala-Pro-Glu-Ala-Ala-Gly-Glu-
Val-Glu-Arg-Leu-Ile-Gly-His-Pro-Leu-Pro-Leu-Arg-Leu-Asp-Ala-Ile-Thr-Gly-Pro-Glu-Glu-Glu-
Gly-Gly-Arg-Leu-Glu-Thr-Ile-Leu-Gly-Trp-Pro-Leu-Ala-Glu-Arg-Thr-Val-Val-Ile-Pro-Ser-Ala-Ile
-Pro-Thr-Asp-Pro-Arg-Asn-Val-Gly-Gly-Asp-Leu-Asp-Pro-Ser-Ser-Ile-Pro-Asp-Lys-Glu-Gln-Ala-
Ile-Ser-Ala-Leu-Pro-Asp-Tyr-Ala-Ser-Gln-Pro-Gly-Lys-Pro-Pro-(Lys-Asp-Glu-Leu)n
说明：该序列的N末端第6位Gly可替换成为Ala、Trp或Leu；该序列的C末端第3位Asp可替换成Gln或Glu；该序列C末端第4位Lys可替换成Arg。n小于等于5大于等于1。并且该序列C末端的氨基酸-(Lys-Asp-Glu-Leu)n可替换成-(Arg-Glu-Asp-Leu-Lys)n，n小于等于5大于等于1。
(4)序列4
Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-Gly-Trp-Glu-Gln-Leu-Glu-Gln-Cys-Gly-Tyr-Pro-Val-
Gln-Arg-Leu-Val-Ala-Leu-Tyr-Leu-Ala-Ala-Arg-Leu-Ser-Trp-Asn-Gln-Val-Asp-Gln-Val-Ile-Arg-
Asn-Ala-Leu-Ala-Ser-Pro-Gly-Ser-Gly-Gly-Asp-Leu-Gly-Glu-Ala-Ile-Arg-Glu-Gln-Pro-Glu-Gln-
Ala-Arg-Leu-Ala-Leu-Thr-Leu-Ala-Ala-Ala-Glu-Ser-Glu-Arg-Phe-Val-Arg-Gln-Gly-Thr-Gly-Asn
-Asp-Glu-Ala-Gly-Ala-Ala-Asn-Ala-Asp-Val-Val-Ser-Leu-Thr-Cys-Pro-Val-Ala-Ala-Gly-Glu-Cys
-Ala-Gly-Pro-Ala-Asp-Ser-Gly-Asp-Ala-Leu-Leu-Glu-Arg-Asn-Tyr-Pro-Thr-Gly-Ala-Glu-Phe-
Leu-Gly-Asp-Gly-Gly-Asp-Val-Ser-Phe-Ser-Thr-Arg-Gly-Thr-Gln-Asn-Trp-Thr-Val-Glu-Arg-Leu-
Leu-Gln-Ala-His-Arg-Gln-Leu-Glu-Glu-Arg-Gly-Tyr-Val-Phe-Val-Gly-Tyr-His-Gly-Thr-Phe-Leu-
Glu-Ala-Ala-Gln-Ser-Ile-Val-Phe-Gly-Gly-Val-Arg-Ala-Arg-Ser-Gln-Asp-Leu-Asp-Ala-Ile-Trp-
Arg-Gly-Phe-Tyr-Ile-Ala-Gly-Asp-Pro-Ala-Leu-Ala-Tyr-Gly-Tyr-Ala-Gln-Asp-Gln-Glu-Pro-Asp-
Ala-Arg-Gly-Arg-Ile-Arg-Asn-Gly-Ala-Leu-Leu-Arg-Val-Tyr-Val-Pro-Arg-Ser-Ser-Leu-Pro-Gly-
Phe-Tyr-Arg-Thr-Ser-Leu-Thr-Leu-Ala-Ala-Pro-Glu-Ala-Ala-Gly-Glu-Val-Glu-Arg-Leu-Ile-Gly-
His-Pro-Leu-Pro-Leu-Arg-Leu-Asp-Ala-Ile-Thr-Gly-Pro-Glu-Glu-Glu-Gly-Gly-Arg-Leu-Glu-Thr-
Ile-Leu-Gly-Trp-Pro-Leu-Ala-Glu-Arg-Thr-Val-Val-Ile-Pro-Ser-Ala-Ile-Pro-Thr-Asp-Pro-Arg-Asn
-Val-Gly-Gly-Asp-Leu-Asp-Pro-Ser-Ser-Ile-Pro-Asp-Lys-Glu-Gln-Ala-Ile-Ser-Ala-Leu-Pro-Asp-
Tyr-Ala-Ser-Gln-Pro-Gly-Lys-Pro-Pro-(Lys-Asp-Glu-Leu)n
说明：该序列的N末端第6位Gly可替换成为Ala、Trp或Leu；该序列的C末端第3位Asp可替换成Gln或Glu；该序列C末端第4位Lys可替换成Arg；n小于等于5大于等于1。并且该序列C末端的氨基酸-(Lys-Asp-Glu-Leu)n可替换成-(Arg-Glu-Asp-Leu-Lys)n，n小于等于5大于等于1。
(5)序列5
Gly-Trp-Glu-Gln-Leu-Glu-Gln-Cys-Gly-Tyr-Pro-Val-Gln-Arg-Leu-Val-Ala-Leu-Tyr-Leu-Ala
-Ala-Arg-Leu-Ser-Trp-Asn-Gln-Val-Asp-Gln-Val-Ile-Arg-Asn-Ala-Leu-Ala-Ser-Pro-Gly-Ser-Gly-
Gly-Asp-Leu-Gly-Glu-Ala-Ile-Arg-Glu-Gln-Pro-Glu-Gln-Ala-Arg-Leu-Ala-Leu-Thr-Leu-Ala-Ala-
Ala-Glu-Ser-Glu-Arg-Phe-Val-Arg-Gln-Gly-Thr-Gly-Asn-Asp-Glu-Ala-Gly-Ala-Ala-Asn-Gly-Pro
-Ala-Asp-Ser-Gly-Asp-Ala-Leu-Leu-Glu-Arg-Asn-Tyr-Pro-Thr-Gly-Ala-Glu-Phe-Leu-Gly-Asp-
Gly-Gly-Asp-Val-Ser-Phe-Ser-Thr-Arg-Gly-Thr-Gln-Asn-Trp-Thr-Val-Glu-Arg-Leu-Leu-Gln-Ala-
His-Arg-Gln-Leu-Glu-Glu-Arg-Gly-Tyr-Val-Phe-Val-Gly-Tyr-His-Gly-Thr-Phe-Leu-Glu-Ala-Ala-
Gln-Ser-Ile-Val-Phe-Gly-Gly-Val-Arg-Ala-Arg-Ser-Gln-Asp-Leu-Asp-Ala-Ile-Trp-Arg-Gly-Phe-
Tyr-Ile-Ala-Gly-Asp-Pro-Ala-Leu-Ala-Tyr-Gly-Tyr-Ala-Gln-Asp-Gln-Glu-Pro-Asp-Ala-Arg-Gly-
Arg-Ile-Arg-Asn-Gly-Ala-Leu-Leu-Arg-Val-Tyr-Val-Pro-Arg-Ser-Ser-Leu-Pro-Gly-Phe-Tyr-Arg-
Thr-Ser-Leu-Thr-Leu-Ala-Ala-Pro-Glu-Ala-Ala-Gly-Glu-Val-Glu-Arg-Leu-Ile-Gly-His-Pro-Leu-
Pro-Leu-Arg-Leu-Asp-Ala-Ile-Thr-Gly-Pro-Glu-Glu-Glu-Gly-Gly-Arg-Leu-Glu-Thr-Ile-Leu-Gly-
Trp-Pro-Leu-Ala-Glu-Arg-Thr-Val-Val-Ile-Pro-Ser-Ala-Ile-Pro-Thr-Asp-Pro-Arg-Asn-Val-Gly-Gly
-Asp-Leu-Asp-Pro-Ser-Ser-Ile-Pro-Asp-Lys-Glu-Gln-Ala-Ile-Ser-Ala-Leu-Pro-Asp-Tyr-Ala-Ser-
Gln-Pro-Gly-Lys-Pro-Pro-Arg-Glu-Asp-Leu-Lys-Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-(Lys
-Asp-Glu-Leu)n
说明：该序列的C末端第9位Gly可替换成为Ala、Trp或Leu；该序列的C末端第3位Asp可替换成Gln或Glu；该序列C末端第4位Lys可替换成Arg；n小于等于5大于等于1。并且该序列C末端的氨基酸-(Lys-Asp-Glu-Leu)n可替换成-(Arg-Glu-Asp-Leu-Lys)n，n小于等于5大于等于1。

Claims (11)

1.一种基因工程重组融和蛋白，其特征是由N末端第一位氨基酸替换成谷氨酸(Glu)的天然人促黄体生成激素释放因子(LHRH)和假单胞菌外毒素A(P E40)的多种突变体串联组成的融合蛋白；以及由N末端第一位氨基酸替换成谷氨酸(Glu)的天然人促黄体生成激素释放因子N末端第6位突变为丙氨酸(Ala)或色氨酸(Trp)或亮氨酸(Leu)的突变体和PE40及其多种突变体串联组成的融合蛋白，该蛋白具有较强的特异性杀伤肿瘤细胞的特性。
2.根据权利要求1，所说的融和蛋白是由由N末端第一位氨基酸替换成谷氨酸(Glu)的天然人促黄体生成激素释放因子或其突变体的蛋白C末端与假单胞菌外毒素A的PE40部分或其突变体的N末端相连，其结构特征为：M-N，M代表N末端第一位氨基酸替换成谷氨酸(Glu)的天然人促黄体生成激素释放因子及其N末端第6位突变为Ala或Trp或Leu的突变体；N代表PE40及其多种突变体，并且M、N之间通过肽键连接。
3.根据权利要求1、2，融和蛋白假单胞菌外毒素A的PE40部分的突变体包括将PE40的C末端5个氨基酸置换为XYEL，其中X代表赖氨酸(Lys)或精氨酸(Arg)，Y代表天冬氨酸(Asp)，谷氨酰胺(Gln)或谷氨酸(Glu)。E代表谷氨酸(Glu)，L代表亮氨酸(Leu)。
4.根据权利要求1、2，将融和蛋白假单胞菌外毒素A PE40的突变体包括对PE40进行缺失365-380位氨基酸成为PE38。
5.根据权利要求4，将PE38的C末端5个氨基酸置换为XYEL，其中X代表赖氨酸(Lys)或精氨酸(Arg)，Y代表天冬氨酸(Asp)，谷氨酰胺(Gln)或谷氨酸(Glu)。E代表谷氨酸(Glu)，L代表亮氨酸(Leu)。
6.根据权利要求1、2，假单胞菌外毒素A PE40的突变体包括对PE40进行缺失253-279和365-380位氨基酸成为PE35。
7.根据权利要求6，将PE35的C末端5个氨基酸置换为XYEL，其中X代表赖氨酸(Lys)或精氨酸(Arg)，Y代表天冬氨酸(Asp)，谷氨酰胺(Gln)或谷氨酸(Glu)。E代表谷氨酸(Glu)，L代表亮氨酸(Leu)。
8.根据权利要求1、2，假单胞菌外毒素A PE40的突变体包括对PE40进行缺失253-279位氨基酸成为PE37。
9根据权利要求8，将PE37的C末端5个氨基酸置换为XYEL，其中X代表赖氨酸(Lys)或精氨酸(Arg)，Y代表天冬氨酸(Asp)，谷氨酰胺(Gln)或谷氨酸(Glu)。E代表谷氨酸(Glu)，L代表亮氨酸(Leu)。
10.将权利要求1-9中组成融和蛋白的两种单一蛋白间连接顺序改变，即M-N变为N-M，同时，在该融和蛋白C末端连上氨基酸序列XYEL或REDLK，即N-M-XYEL或N-M-REDLK，其中X代表赖氨酸(Lys)或精氨酸(Arg)，Y代表天冬氨酸(Asp)，谷氨酰胺(Gln)或谷氨酸(Glu)。E代表谷氨酸(Glu)，L代表亮氨酸(Leu)，R代表精氨酸(Arg)，D代表天冬氨酸(Asp)，K代表赖氨酸(Lys)。
11.将权利要求3、5、7、9、10中融和蛋白序列C末端XYEL或REDLK片段重复串连2-5次，即M-N-(XYEL)n、M-N-(REDLK)n、N-M-(XYEL)n、N-M-(REDLK)n，其中n≤5。其中X代表赖氨酸(Lys)或精氨酸(Arg)，Y代表天冬氨酸(Asp)，谷氨酰胺(Gln)或谷氨酸(Glu)。E代表谷氨酸(Glu)，L代表亮氨酸(Leu)，R代表精氨酸(Arg)，D代表天冬氨酸(Asp)，K代表赖氨酸(Lys)。
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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006045233A1 (fr) * 2004-10-26 2006-05-04 Beijing Northland Biotech Co., Ltd. PROTÉINE MARQUÉE AU TECHNÉTIUM-99M ET COMPRENANT LA GnRH OU LA GnRH MUTANTE
CN101433713B (zh) * 2007-11-15 2011-10-12 北京诺思兰德生物技术股份有限公司 GnRH-PE突变体融合蛋白及其用途
CN101195822B (zh) * 2006-12-08 2012-08-08 湖南康都制药有限公司 高亲和性靶向融合蛋白
WO2015143581A1 (zh) * 2014-03-24 2015-10-01 北京博翱泰生物技术有限公司 靶特异性双突变体融合蛋白质及其制备工艺

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006045233A1 (fr) * 2004-10-26 2006-05-04 Beijing Northland Biotech Co., Ltd. PROTÉINE MARQUÉE AU TECHNÉTIUM-99M ET COMPRENANT LA GnRH OU LA GnRH MUTANTE
CN101195822B (zh) * 2006-12-08 2012-08-08 湖南康都制药有限公司 高亲和性靶向融合蛋白
CN101433713B (zh) * 2007-11-15 2011-10-12 北京诺思兰德生物技术股份有限公司 GnRH-PE突变体融合蛋白及其用途
WO2015143581A1 (zh) * 2014-03-24 2015-10-01 北京博翱泰生物技术有限公司 靶特异性双突变体融合蛋白质及其制备工艺

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