具体实施方式
下面通过实施例和对比例进一步说明本发明,这些实施例只是用于说明本发明,本发明不限于以下实施例。凡是对本发明技术方案进行修改或者等同替换,而不脱离本发明技术方案的范围,均应涵盖在本发明的保护范围中。
以下实施例中所采用的FAP抗原氨基酸序列如下:
LEVDEITLWYKMILPPEFDRSKKYPLLIQVYGGPCSQSVRSVFAVNWISYLASKEGMVIALVDGRGTAFQGDKFLY
AVYRKLGVYEVEDQITAVRKFIEMGFIDEKRIAIWGWSYGGYISSLALASGTGLFKCGIAVAPVSSWEYYASIYTERFMG
LPTKDDNLKHYKNSTVMARAEYFRNVDYLLIHGTADDNVHFQNSAQIAKALVNAEVDFQAMWYSDQNHGLSGQSTNHLYT
HMTHFLKQCFSLLD,如SEQ ID NO:1所示。
一些实施例中,该FAP抗原可以来源于人FAP蛋白胞外区,该人FAP蛋白的氨基酸序列如下:
MKTWVKVLSGVATSAVLALLVMCIVLRPSRVHNSEENTMTALTLQDILNGTFSYKTFFPNWISGQEYLHKSAENNIVLYNIETGQSYTILSNRTTKSVNASGYGLSPDRQFIYLESDYSKLWRYSYTATYYIYDLSNGEFVRGNELPRPIQYLCWSPVGSKLAYVYQNNIYLKQRPGDPPFQITFNGRENKIFNGIPDWVYEEEMLATKYALWWSPNGKFLAYVEFNDTDIPVIAYSYYGDGQYPRTINIPYPKAGAKNPVVRVFIIDTTYPAYVGPQEVPVPAMIASSDYYFSWLTWVTDERVCLQWLKRIQNVSVLSICDFREDWQTWDCPKFFVSTPVFSYDAISYYKIFSDKDGYKHIHYIKDTVENAIQITSGKWEAINIFRVTQDSLFYSSNEFEDYPGRRNIYRISIGSYPPSKKCVTCHLRKERCQYYTASFSDYAKYYALICYGPGIPISTLHDGRTDQEIKILEENKELEDSLKNIQLPKEEIKKLEVDEITLWYKMILPPEFDRSKKYPLLIQVYGGPCSQSVRSVFAVNWISYLASKEGMVIALVDGRGTAFQGDKFLYAVYRKLGVYEVEDQITAVRKFIEMGFIDEKRIAIWGWSYGGYISSLALASGTGLFKCGIAVAPVSSWEYYASIYTERFMGLPTKDDNLKHYKNSTVMARAEYFRNVDYLLIHGTADDNVHFQNSAQIAKALVNAEVDFQAMWYSDQNHGLSGQSTNHLYTHMTHFLKQCFSLLD,如SEQID NO:2所示,上述FAP抗原的氨基酸序列为其自碳末端的250个氨基酸序列。
上述FAP抗原在制备预防或治疗心力衰竭药物中的应用,心力衰竭是指缺血性心肌病、扩张性心肌病、肥厚性心肌病和高血压性心肌病所致的慢性进行性、以成纤维细胞活化和增生为病理基础的心力衰竭。
一些实施例中,药物可为肽类疫苗。
本发明的肽类疫苗包括上述FAP抗原,还包括能够特异性靶向免疫***至少一种抗原呈递细胞的纳米外壳,且FAP抗原包裹于其内部。
一些实施例中,纳米外壳由脂质纳米颗粒构成,且其表面以脂质镶嵌的方式连接具有靶向结合至少一种抗原呈递细胞且以壳聚糖为基础的特异性靶标。
一些实施例中,抗原呈递细胞可以为巨噬细胞或树突状细胞,特异性靶标可以为甘露糖。
一些实施例中,肽类疫苗还包括免疫佐剂CpG。
以下实施例1示例性地制备一种以活化成纤维FAP为基础的肽类疫苗,FAP抗原为上海生工生物工程有限公司合成生产,其他未注明制备方法的原料均为市售商品。
实施例1
以活化成纤维FAP为基础的肽类疫苗,制备方法如下:
第一步:先通过薄膜法制备脂质体纳米颗粒,在旋蒸的过程中将胆固醇-PEG连接的甘露糖按质量比为1:1000加入到上述脂质纳米颗粒的母液中旋蒸,得到甘露糖功能化的脂质薄膜作为纳米外壳;
第二步:免疫佐剂和FAP抗原加入到装有上述脂质薄膜的旋蒸瓶内进行水化,经过超声震荡处理得到。
实施例2
将实施例1制备的肽类疫苗通过静脉注射入健康小鼠和有心力衰竭症状的小鼠体内,注射量按脂质纳米外壳的量为1mg/每只小鼠,经过一段时间观察和对照,发现注射该肽类疫苗后,小鼠的抗心力衰竭免疫效果显著。
上述对实施例的描述是为了便于该技术领域的普通技术人员能理解和使用本发明。熟悉本领域技术人员显然可以容易的对这些实施例做出各种修改,并把在此说明的一般原理应用到其他实施例中,而不必经过创造性的劳动。因此,本发明不限于上述实施例。本领域技术人员根据本发明的原理,不脱离本发明的范畴所做出的改进和修改都应该在本发明的保护范围之内。
序列表
<110> 复旦大学附属中山医院
<120> 以活化成纤维FAP为基础的肽类疫苗及其制备方法
<141> 2021-04-27
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 250
<212> PRT
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 1
Leu Glu Val Asp Glu Ile Thr Leu Trp Tyr Lys Met Ile Leu Pro Pro
1 5 10 15
Glu Phe Asp Arg Ser Lys Lys Tyr Pro Leu Leu Ile Gln Val Tyr Gly
20 25 30
Gly Pro Cys Ser Gln Ser Val Arg Ser Val Phe Ala Val Asn Trp Ile
35 40 45
Ser Tyr Leu Ala Ser Lys Glu Gly Met Val Ile Ala Leu Val Asp Gly
50 55 60
Arg Gly Thr Ala Phe Gln Gly Asp Lys Phe Leu Tyr Ala Val Tyr Arg
65 70 75 80
Lys Leu Gly Val Tyr Glu Val Glu Asp Gln Ile Thr Ala Val Arg Lys
85 90 95
Phe Ile Glu Met Gly Phe Ile Asp Glu Lys Arg Ile Ala Ile Trp Gly
100 105 110
Trp Ser Tyr Gly Gly Tyr Ile Ser Ser Leu Ala Leu Ala Ser Gly Thr
115 120 125
Gly Leu Phe Lys Cys Gly Ile Ala Val Ala Pro Val Ser Ser Trp Glu
130 135 140
Tyr Tyr Ala Ser Ile Tyr Thr Glu Arg Phe Met Gly Leu Pro Thr Lys
145 150 155 160
Asp Asp Asn Leu Lys His Tyr Lys Asn Ser Thr Val Met Ala Arg Ala
165 170 175
Glu Tyr Phe Arg Asn Val Asp Tyr Leu Leu Ile His Gly Thr Ala Asp
180 185 190
Asp Asn Val His Phe Gln Asn Ser Ala Gln Ile Ala Lys Ala Leu Val
195 200 205
Asn Ala Glu Val Asp Phe Gln Ala Met Trp Tyr Ser Asp Gln Asn His
210 215 220
Gly Leu Ser Gly Gln Ser Thr Asn His Leu Tyr Thr His Met Thr His
225 230 235 240
Phe Leu Lys Gln Cys Phe Ser Leu Leu Asp
245 250
<210> 2
<211> 745
<212> PRT
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 2
Met Lys Thr Trp Val Lys Val Leu Ser Gly Val Ala Thr Ser Ala Val
1 5 10 15
Leu Ala Leu Leu Val Met Cys Ile Val Leu Arg Pro Ser Arg Val His
20 25 30
Asn Ser Glu Glu Asn Thr Met Thr Ala Leu Thr Leu Gln Asp Ile Leu
35 40 45
Asn Gly Thr Phe Ser Tyr Lys Thr Phe Phe Pro Asn Trp Ile Ser Gly
50 55 60
Gln Glu Tyr Leu His Lys Ser Ala Glu Asn Asn Ile Val Leu Tyr Asn
65 70 75 80
Ile Glu Thr Gly Gln Ser Tyr Thr Ile Leu Ser Asn Arg Thr Thr Lys
85 90 95
Ser Val Asn Ala Ser Gly Tyr Gly Leu Ser Pro Asp Arg Gln Phe Ile
100 105 110
Tyr Leu Glu Ser Asp Tyr Ser Lys Leu Trp Arg Tyr Ser Tyr Thr Ala
115 120 125
Thr Tyr Tyr Ile Tyr Asp Leu Ser Asn Gly Glu Phe Val Arg Gly Asn
130 135 140
Glu Leu Pro Arg Pro Ile Gln Tyr Leu Cys Trp Ser Pro Val Gly Ser
145 150 155 160
Lys Leu Ala Tyr Val Tyr Gln Asn Asn Ile Tyr Leu Lys Gln Arg Pro
165 170 175
Gly Asp Pro Pro Phe Gln Ile Thr Phe Asn Gly Arg Glu Asn Lys Ile
180 185 190
Phe Asn Gly Ile Pro Asp Trp Val Tyr Glu Glu Glu Met Leu Ala Thr
195 200 205
Lys Tyr Ala Leu Trp Trp Ser Pro Asn Gly Lys Phe Leu Ala Tyr Val
210 215 220
Glu Phe Asn Asp Thr Asp Ile Pro Val Ile Ala Tyr Ser Tyr Tyr Gly
225 230 235 240
Asp Gly Gln Tyr Pro Arg Thr Ile Asn Ile Pro Tyr Pro Lys Ala Gly
245 250 255
Ala Lys Asn Pro Val Val Arg Val Phe Ile Ile Asp Thr Thr Tyr Pro
260 265 270
Ala Tyr Val Gly Pro Gln Glu Val Pro Val Pro Ala Met Ile Ala Ser
275 280 285
Ser Asp Tyr Tyr Phe Ser Trp Leu Thr Trp Val Thr Asp Glu Arg Val
290 295 300
Cys Leu Gln Trp Leu Lys Arg Ile Gln Asn Val Ser Val Leu Ser Ile
305 310 315 320
Cys Asp Phe Arg Glu Asp Trp Gln Thr Trp Asp Cys Pro Lys Phe Phe
325 330 335
Val Ser Thr Pro Val Phe Ser Tyr Asp Ala Ile Ser Tyr Tyr Lys Ile
340 345 350
Phe Ser Asp Lys Asp Gly Tyr Lys His Ile His Tyr Ile Lys Asp Thr
355 360 365
Val Glu Asn Ala Ile Gln Ile Thr Ser Gly Lys Trp Glu Ala Ile Asn
370 375 380
Ile Phe Arg Val Thr Gln Asp Ser Leu Phe Tyr Ser Ser Asn Glu Phe
385 390 395 400
Glu Asp Tyr Pro Gly Arg Arg Asn Ile Tyr Arg Ile Ser Ile Gly Ser
405 410 415
Tyr Pro Pro Ser Lys Lys Cys Val Thr Cys His Leu Arg Lys Glu Arg
420 425 430
Cys Gln Tyr Tyr Thr Ala Ser Phe Ser Asp Tyr Ala Lys Tyr Tyr Ala
435 440 445
Leu Ile Cys Tyr Gly Pro Gly Ile Pro Ile Ser Thr Leu His Asp Gly
450 455 460
Arg Thr Asp Gln Glu Ile Lys Ile Leu Glu Glu Asn Lys Glu Leu Glu
465 470 475 480
Asp Ser Leu Lys Asn Ile Gln Leu Pro Lys Glu Glu Ile Lys Lys Leu
485 490 495
Glu Val Asp Glu Ile Thr Leu Trp Tyr Lys Met Ile Leu Pro Pro Glu
500 505 510
Phe Asp Arg Ser Lys Lys Tyr Pro Leu Leu Ile Gln Val Tyr Gly Gly
515 520 525
Pro Cys Ser Gln Ser Val Arg Ser Val Phe Ala Val Asn Trp Ile Ser
530 535 540
Tyr Leu Ala Ser Lys Glu Gly Met Val Ile Ala Leu Val Asp Gly Arg
545 550 555 560
Gly Thr Ala Phe Gln Gly Asp Lys Phe Leu Tyr Ala Val Tyr Arg Lys
565 570 575
Leu Gly Val Tyr Glu Val Glu Asp Gln Ile Thr Ala Val Arg Lys Phe
580 585 590
Ile Glu Met Gly Phe Ile Asp Glu Lys Arg Ile Ala Ile Trp Gly Trp
595 600 605
Ser Tyr Gly Gly Tyr Ile Ser Ser Leu Ala Leu Ala Ser Gly Thr Gly
610 615 620
Leu Phe Lys Cys Gly Ile Ala Val Ala Pro Val Ser Ser Trp Glu Tyr
625 630 635 640
Tyr Ala Ser Ile Tyr Thr Glu Arg Phe Met Gly Leu Pro Thr Lys Asp
645 650 655
Asp Asn Leu Lys His Tyr Lys Asn Ser Thr Val Met Ala Arg Ala Glu
660 665 670
Tyr Phe Arg Asn Val Asp Tyr Leu Leu Ile His Gly Thr Ala Asp Asp
675 680 685
Asn Val His Phe Gln Asn Ser Ala Gln Ile Ala Lys Ala Leu Val Asn
690 695 700
Ala Glu Val Asp Phe Gln Ala Met Trp Tyr Ser Asp Gln Asn His Gly
705 710 715 720
Leu Ser Gly Gln Ser Thr Asn His Leu Tyr Thr His Met Thr His Phe
725 730 735
Leu Lys Gln Cys Phe Ser Leu Leu Asp
740 745